DNA encoding C5A receptor antagonists having substantially no agonist activity and methods of expressing same

ABSTRACT

Disclosed are polypeptide analogues of human C5a which are C5a receptor antagonists that exhibit substantially no analphylatoxin or agonist activity, and derivatives of the analogues, and dimeric forms of the analogues or derivatives. DNA molecules encoding the polypeptides and methods of making the analogues are also provided. Pharmaceutical formulations containing a C5a analogue, are used therapeutically in the treatment of C5a-mediated diseases and inflammatory conditions in mammals, and prophylactically to prevent or reduce inflammation caused by an event which causes inflammation or aggravates an existing inflammatory condition, respectively. Further disclosed are antibodies specific to the C5a analogues, derivatives thereof, and dimers of the analogues and derivatives which exhibit substantially no cross-reactivity with human C5a. The antibodies are used to detect or quantify circulating C5a analogue or derivative, as well as to modify, e.g., neutralize, the activity of the C5a receptor antagonist in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application ofPCT-IB94-00359, filed Nov. 16, 1994, which is a continuation-in-part ofU.S. patent application Ser. No. 08/162,591, filed Dec. 6, 1993, nowabandoned, the contents of which are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to the field of immunology, and morespecifically to the treatment: of complement-mediated diseases andinflammatory conditions in mammals.

BACKGROUND OF THE INVENTION

Inflammation is a localized, protective event, elicited by injury, whichserves to destroy, dilute or wall off both injurious agents and theinjured tissues. It involves a complex series of events, includingdilation of arteries, capillaries and venules, with increased vascularpermeability, increased blood flow, and exudation of fluids and plasmaproteins. These processes are often rapidly followed by adhesion ofleukocytes to the vascular endothelium, with subsequent influx of thecells into the surrounding tissue.

The complement system, a major immunological defense mechanism againstforeign substances, has been shown to influence each of the factors thatcomprise the inflammatory response. In general, complement comprises aset of proteins that work to eliminate microorganisms, and otherantigens from tissues and blood. This task is achieved either bycomplement components alone or in cooperation with antibodies or withcells that express complement receptors. More specifically, the systemconsists of about 30 plasma proteins, their corresponding cellularreceptors and several membrane regulatory proteins. Kinoshita,Immunology , 12:291-300 (1991). Activation of the complement system by,for example, antigen-antibody complexes or bacterial surface structures,triggers an amplification cascade of proteolytic cleavage and proteinassembly events of the complement components, which ultimately leads tothe destruction and final elimination of the foreign body.Muller-Eberhard, Annu. Rev. Biochem. 57:321-347 (1988).

Several biologically active peptides are generated by the activation ofthe complement system. C5a, a glycoprotein containing 74 amino acids andhaving an M_(r) of about 11,000, is generated by the proteolyticcleavage of the N-terminal end of C5, the fifth component of complement,by C5 convertase. Nilsson et al., J. Immunol. 114:815-822 (1975). Thebiological properties of C5a extend across a multitude of cells andtissues involved in both acute and chronic inflammatory processes.Hugli, CRC Crit. Rev. Immunol. 1:321-366 (1981). Mary of theseproperties are immunologically beneficial. C5a has been found to mediatehost defense mechanisms in response to various pathological conditions.C5a participates in a wide variety of specific biologic functionscommonly associated with the inflammatory response, such as smoothmuscle contraction, an increase in vascular permeability, wheal andflare generation when injected into human skin, histamine release frommast cells, and induction of the oxidative burst and lysosomal enzymerelease from polymorphonuclear leukocytes (PMNLs). C5a stimulatesmeasurable responses from every circulating white blood cell includingbasophils, eosinophils, monocytes, and neutrophils. Hugli, supra.;Bautsch et al., Immunobiol. 185:41-52 (1992). C5a has further been foundto be a potent chemoattractant. Fernandez et al., J. Immunol.120:109-115 (1978). This protein is a pivotal stimulus to the attractionof PMNLs such as phagocytic cells to the site of inflammation.

Complement is beneficial when directed against an appropriate targetsuch as invading microorganisms or tumor cells, but has clear pathogenicpotential if activated inappropriately. For instance, theanaphylatoxins, e.g., C5a, have been implicated as causative oraggravating factors in the pathogenesis of several inflammatory diseasessuch as adult respiratory distress syndrome and rheumatoid arthritis.Bautsch et al., Biochem. J. 288:261-266 (1992); Haslett et al., J.Immunol. 142:3510-3517 (1989). In particular, the aberrant presence ofC5a in tissue has been detected in patients afflicted with rheumatoidarthritis, osteoarthritis, psoriasis and noncardiac pulmonary edema. C5ahas been found to be a principal inflammatory mediator produced bycomplement activation by virtue of additional activities that includerecruitment and stimulation of inflammatory leukocytes and augmentationof antibody production. See Mollison et al., Proc. Natl. Acad. Sci. USA86:292-296 (1989).

The in vivo or pharmacologic control of inflammation is presumed to bedependent on the modulation of chemotaxis. Three levels at whichinhibition can occur have been recognized. These are (1) suppression ofthe leukocytic response to chemotactic stimuli; (2) prevention ofchemotaxin generation; and (3) inactivation of the chemotaxins. Inaddition, because C5a exerts its various functions by binding to aspecific C5a receptor found in the membrane of several human cells suchas neutrophils, eosinophils and monocyte-derived cells, the inhibitionof C5a-mediated chemotaxis, and in particular, the design of C5areceptor antagonists have attracted considerable attention.

U.S. Pat. No. 4,772,584 to Cleary et al. discloses polypeptides isolatedfrom group A streptococci which inhibit the binding of C5a to PMNLs bycleaving a six amino acid peptide from the C-terminus of C5a. U.S. Pat.No. 4,692,511 to Hahn teaches polypeptide receptor antagonists to C5awhich contain an essential core tetrapeptide Tyr-Asp-Gly-Ala (SEQ IDNO. 1) or Asp-Gly-Ala-Tyr (SEQ ID NO. 2), or core tripeptide Asp-Gly-Alawhich display C5a blocking activity.

U.S. Pat. No. 5,190,922, WO 90/09162 and WO 92/11858 to AbbottLaboratories disclose various oligopeptides which bind to C5a receptorsand purportedly modulate anaphylatoxin activity. However, several ofthese molecules have been shown to retain significant agonist activity.See Mollison et al., "C5a Structural Requirements for NeutrophilReceptor Interaction," in Progress in Inflammation Research and Therapy,Birkhauser Verlag, Basel (1991) at pages 17-21; Kawai et al., J. Med.Chem. 35:220-223 (1992); Kawai et al., J. Med. Chem. 34:2068-2071(1991); and Or et al., J. Med. Chem. 35:402-406 (1992). Hence, thereremains a strong need for a potent and therapeutically effective C5areceptor antagonist which is substantially void of agonist activity.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to polypeptidicanalogues of human C5a which are C5a receptor antagonists and exhibitsubstantially no anaphylatoxin or agonist activity, derivatives of theanalogues, and dimeric forms of the analogues and derivatives. DNAmolecules encoding the polypeptides, (i.e., the analogues andderivatives thereof) plasmids, vectors and host cells transformed withthe DNA molecules, and methods of preparing the C5a analogues are alsoprovided.

Pharmaceutical formulations containing a C5a analogue, derivative ordimer thereof are advantageously used in methods for the treatment ofC5a-mediated inflammatory conditions and diseases in mammals, and as aprophylactic to prevent such inflammation.

Another aspect of the present invention is directed to antibodiesspecific to the C5a analogue and derivatives thereof, which exhibitsubstantially no cross-reactivity with human C5a. The antibodies areused to detect or quantify circulating C5a analogues and derivatives insubjects (previously administered with same) as well as to modify, e.g.,neutralize, the activity of the C5a analogues and derivatives in vivo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow diagram that illustrates the synthesis of a syntheticgene encoding human C5a via oligonucleotide coupling; and

FIG. 2 is a plasmid map of pB-6/C5a.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The C5a polypeptide analogues of the present invention are C5a receptorantagonists which have substantially no agonist activity. The term "C5areceptor" is understood in the art as referring to the sites on thesurfaces of human blood cells such as PMNLs and monocytic cells, towhich C5a, its degradation product C5a-desArg, and the instantantagonists bind. See, for example, U.S. Pat. No. 5,177,190 andOppermann et al, J. Immunol. 151(7):3785-3794 (1993). C5a is convertedenzymatically to C5a-desArg in human serum by a carboxypeptidase B-likeenzyme, and is the major physiological end product in man. Chenoweth etal., Mol. Immunol. 17:151-161 (1980).

By the term "antagonist," it is meant that the instantly disclosedpolypeptides are inhibitors of C5a. That is, they interfere with thebinding of C5a to the C5a receptor. While not intending to be bound byany particular theory, Applicants believe that the C5a analogues arecompetitive inhibitors of C5a in that they compete with C5a for bindingto the C5a receptor.

The antagonism of the instant C5a analogues may be quantified as an IC₅₀in the calcium rise assay disclosed in Agents and Actions 21:375-378(1987), described in detail in Example 7. The IC₅₀ is defined as theconcentration of C5a analogue which inhibits 50% of the intracellularmobilization of calcium ions by the PMNLs bearing the C5a receptor,after a challenge dose with 100 pM human C5a. The C5a receptorantagonists of the present invention exhibit an IC₅₀ of no greater thanabout 2.0×10⁻⁶ M in the calcium rise assay disclosed in Seligmann et al.

By the phrases "substantially no anaphylatoxin activity" or"substantially no agonist activity," it is meant that the binding of theC5a analogue (hereinafter used interchangeably with C5a receptorantagonist) to the receptor does not result in an endogenous signaltransduction event ultimately resulting in the physiological responsescommonly associated with anaphylatoxin-induced inflammation caused bybinding of C5a to its receptor, such as activation of phagocytic cells,smooth muscle contraction, increase in vascular permeability, andexcessive production of inflammatory mediators, e.g., histamines,prostaglandins, thromboxatnes, leukotrienes, interleukin (IL)-1, IL-6and IL-8. See Hugli et al., CRC Crit. Rev. Immunol. 1:321-326 (1981) andPCT WO 92/10205. A quantitative measure of this property may also beobtained using the calcium rise assay disclosed in Seligmann et al.,supra, also described in Example 7. EC₅₀ is a measure of agonisticactivity. For purposes of the present invention, the EC₅₀ value is thatconcentration of C5a analogue which produces 50% of the maximum responsecaused by that same C5a analogue. Applicants have not detected agonistactivity of the instant C5a analogues up to a concentration of at Leastabout 8.0×10⁻⁷ M, and preferably at least about 3.0×10⁻⁶ M in the samecalcium rise assay. The C5a analogues of the present invention are thosefor which the EC₅₀ is not measurable in the Seligmann calcium rise assayup to C5a analogue concentrations of at least about 8.0×10⁻⁷ M, andpreferably at least about 3.0×10⁻⁶ M, since no response can be detectedin the assay.

C5a is a 74-amino acid polypeptide, the sequence of which has beendisclosed in Fernandez et al., J. Biol. Chem. 253:6955-6964 (1978).Synthetic genes, constructed based upon the deduced nucleotidesequences, are disclosed in Mandecki et al, Proc. Nat'l Acad. Sci. USA82:3543-3547 (1985) and U.S. Pat. No. 4,937,189 to Davidow et al. Theamino acid sequence of C5a disclosed in Fernandez, and the correspondingsynthetic nucleotide sequence disclosed in Davidow et al. are set forthin Table 1, below.

                                      TABLE 1                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                     __________________________________________________________________________

Applicants have unexpectedly and surprisingly discovered that certainanalogues of human C5a, produced by mutagenizing the portion of asynthetic C5a gene encoding the C-terminal region, i.e., amino acids64-74, of human C5a (hereinafter used interchangeably with "C5a(1-74)"),have dramatically different properties than C5a. That is, they exhibitexcellent antagonistic properties and substantially no agonist activity.Specifically, the C5a analogues of the present invention are defined interms of two modifications or mutations to the C-terminal region##STR7## (amino acids 64-74 of C5a (1-74)). First, it is truncated atleast to Leu (72); i.e., by removing the Gly (73) and Arg (74) residues.Second, at least one cysteine is substituted in the region, providedthat the C-terminal amino acid of the polypeptide (i.e., the C-terminus)is cysteine, and that the thiol (SH) group of the C-terminal cysteine isin reduced form (i.e., has a free thiol group), or is in a form capableof spontaneously converting or being readily converted into a free thiolgroup.

In a preferred embodiment, from 2 to 6 of the most C-terminal aminoacids are truncated from C5a (1-74). Thus, in the case where theN-terminal 63 amino acid region is kept intact and only one cysteine issubstituted, the respective corresponding embodiments may be designatedas follows: C5a (1-72, Leu72Cys), C5a. (1-71, Gln71Cys), C5a (1-70,Met70Cys), C5a (1-69, Asp69Cys) and C5a (1-68, Lys68Cys). In a morepreferred embodiment, the C-terminal region is truncated to andincluding Met70, Gln71or Leu72, which correspond to the three formerdesignated embodiments. An even more preferred embodiment is C5a (1-71,Gln71Cys).

The C-terminal region can be further truncated N-terminally to Asn 64,which would correspond to the representative designated embodiments C5a(1-67, His67Cys), C5a (1-66, Ser66Cys), C5a (1-65, Ile65Cys) and 65a(1-64, Asn64Cys), provided that the resultant C5a analogue exhibits theforementioned requisite antagonist property (an IC₅₀ of no greater thanabout 2.0×10⁻⁶ M) and substantially no anaphylatoxin or agonist activity(a non-measurable EC₅₀ up to C5a analogue concentrations of at least3.0×10⁻⁶ M). Those skilled in the art would understand that "analogues"of human C5a do not include antibodies specific to C5a or to sites onthe C5a receptor.

Derivatives of the human C5a analogues as described herein are includedwithin the scope of the present invention. These include modificationssuch as point mutations, substitutions, additions and deletions in theN-terminal 63 amino acid region (amino acids 1-63 of C5a(1-74)), Carneyet al., Protein Science 2:1391-1399 (1993), and further amino acidsubstitutions in the thus-mutagenized C-terminal region. The type andextent of the modifications are generally not important, so long as theresultant derivative remains a C5a receptor antagonist withsubstantially no agonist activity, both as defined above. For example,the Cys27 residue in the N-terminal region of C5a (1-74) can be changed,e.g., to a serine residue, in order to minimize complications duringrefolding. Thus, in a more preferred embodiment, the C5a analoguederivative is designated C5a(1-71, Cys27Ser, Gln71 Cys). Also, theN-terminus may be changed to a Methionine residue, either bysubstitution or addition, to allow for expression of a C5aanalogue-encoding gene in various host cells such as E. coli. Any C5aanalogue of the present invention produced in E. coli will have a Metresidue as its N-terminus, as explained in Example 1 and shown in Table3 (Example 3, below).

N-terminal substitutions also occur in cases where the human C5a analogsare expressed in various host cells, e.g., E. coli, as a fusion protein,and then isolated by cleaving the fusion protein at a convenient site.For example, cleavage of a human C5a analogue from its fusion proteinpartner linked via an hydroxylamine sensitive linkage, results in thesubstitution of a glycine (Gly) residue for the native human C5athreonine (Thr) N-terminus. Thus, in another more preferred embodiment,the C5a analogue derivative is designated C5a (1-71, Thr1Gly, Cys27Ser,Gln71Cys).

An example of a further modification of the C-terminal region is thesubstitution of a phenylalaniine residue for the native histidine atposition 67 of C5a(1-74). Thus, in a most preferred embodiment, the C5aanalogue derivative is designated C5a (1-71, Thr1Met, Cys27Ser,His67Phe, Gln71Cys).

The C5a analogues (hereinafter referring collectively to the analoguesand derivatives thereof) of the present invention can be prepared vianumerous procedures standard in the art. For instance, they may beprepared via direct chemical synthesis. They may also be prepared byexpression of DNA molecules, i.e., synthetic genes, encoding thepolypeptides in suitable host cells. These DNA molecules, deducible fromthe amino acid sequences of the C5a analogues, in turn may be preparedvia known techniques. The DNAs may be synthesized chemically asdisclosed in Narang, Tetrahedron 393:3-22(1983); EPA 146,785; Mandeckiet al., Proc. Natl. Acad. Sci. USA 82:3543-3547; and (1985) (disclosingthe chemical synthesis of a gene encoding C5a). Fragments of the DNAmolecules may be prepared chemically, which then are linked togetherenzymatically. See Volume 1, Chapter 8 of Current Protocols in MolecularBiology, Ausubel et al. (Eds.), Wiley, N.Y. (1990).

DNAs encoding the C5a analogues of the present invention can also beprepared by mutagenesis of known synthetic or natural genes encodingC5a, such as those disclosed in Fernandez, Mandecki Davidson, forexample. See Ausubel et al., supra.; Volume II, Chapter 15 of Maniatiset al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, N.Y.(1989); and Mollison et al., Proc. Natl. Acad. Sci. USA 86:292-296(1989). Further, the DNAs may be prepared via PCR techniques. PCRProtocols, Innis et al. (Eds.), Academic Press, San Diego, Calif.(1990).

The DNA molecules encoding the C5a analogues of the present inventionare operably linked to known regulatory sequences, e.g., promoter,enhancer, 3'-untranslated sequences, and 5' translated sequences, e.g.,signal and leader sequences, and then transformed into host cellscapable of expressing the genes, in accordance with standard techniques.Then, the transformed host cells are cultured under conditions suitablefor expression of the antagonist encoding gene. Representative hostcells include prokaryotes such as E. coli and Bacillus, e.g., B.subtilis; and eukaryotes such as filmentous fungi, e.g., Aspergillusniger; yeast, e.g., Saccharomyces cerevisiae, Pichia pastoris andYarrowia lipolytica; baculovirus/insect cell cultures (Summers et al., AManual of Methods for Baculoivirus Vectors and Insect Cell CultureProcedures, Texas Agricultural Experiment Station (1987)); mammaliancell lines; and plants (J. Vandekerckhove et al., BIO/TECHNOLOGY7:929-932 (1989)).

In general, the procedures for expression of C5a in E. coli areapplicable to C5a analogue gene expression. See, Mandecki, Proc. Natl.Acad. Sci. USA 82:3543-3547 (1985); Mollison et al., Proc. Natl. Acad.Sci. USA 86:292-296 (1989); and Bautsch et al., Immunobiol. 185:41-52(1992). The choice of suitable regulatory sequences such as promoter(e.g., T7 polymerase, UV5-D, trp or lac), ribosome binding site, as wellas suitable plasmid vectors containing transcriptional stop sites, e.g.,pKK223-2, are within the level of skill in the art. To optimizeexpression in E. coli, the DNA molecule should be synthesized using E.coli-preferred codons as disclosed in Guoy et al., Nucleic Acids Res.10:7055-7074 (1982), and to allow for several restriction endonucleasesites to facilitate characterization of the synthesized DNA and possiblymutagenesis of the DNA sequence. This approach allows for directexpression of the C5a analogue, by introducing an ATG initiation codonfor protein synthesis directly and immediately upstream of the tripletcoding for the first amino acid of the polypeptide. Further, E. colistrains, e.g., ion, which are deficient in one of several proteasespresent in wild-type cells offer the advantage of increased yield ofprotein. Franke et al., Meth. Enzymol. 162:653-658 (1988).

Expression of the C5a analogue genes in hosts such as E. coli may alsobe enhanced by expressing the genes in the form of a fusion protein.Methods; of preparing such fusion proteins using chemically andenzymatically cleavable linkages are known in the art. See, e.g., Smith,in "Methods in Molecular Biology," Vol. 3, "New Protein Techniques", pp.57-70 and 71-88 (1984), and Van Heeke et al., Protein Expression andPurification 4:265-74 (1993). Fusion partners, in general, are thosegenes (or fragments thereof) which are highly expressed in the host,e.g., E. coli. In the case of E. coli, suitable fusion partners includeendogenous E. coli genes and synthetic genes, e.g., containing E.coli-preferred codons. In addition, the smaller the size of the fusionpartner, the better the yield of the C5a analogue, or derivativethereof. In a preferred embodiment for expression in E. coli, the fusionpartner is a 159-nucleotide DNA encoding a 53 amino acid fragment of ahybrid of the proteins human interleukin 1 beta and interleukin 1receptor antagonist (Dinarello, Blood 77:1627-52 (1991)), which DNAcontains E. coli-preferred codons, and a 24 nucleotide fragment encodingan 8 amino acid linker containing an enterokinase cleavage site and ahydroxylamine sensitive site (Asn-Gly). Those skilled in the art candetermine the appropriate size of a fusion partner in accordance withstandard techniques (e.g. by determining expression titers of C5aanalogue fusion proteins by systematically varying the size (length) ofthe fusion partner).

In general, the C5a analogue-encoding synthetic genes can be expressedin yeast by following known procedures. See, for example, Romanos etal., Yeast 8:423-488 (1992); Section IV of Goeddel (Ed.), Seth. Enzymol.185:231-484 (1990); Davidow et al., supra. and U.S. Pat. No. 4,775,622.To optimize expression in yeast, the DNA molecule should be preparedusing yeast-preferred codons, particularly to avoid Arg-Arg pairs whichare targets for endogenous KEX2 proteases. The use of glutamine, asopposed to methionine, as the N-terminus, facilitates proteolyticcleavage from the signal sequence, e.g., alpha factor signal sequence.It is further preferred to eliminate any potential glycosylation sitessuch as the asparagine at position 64 of various embodiments of theinstant C5a receptor antagonists.

Expression of a C5a analogue-encoding gene of the present invention inmammalian cells can be performed in accordance with known procedures.See Chapter 16, "Expression of Cloned Genes in Mammalian Cells," inManiatis et al., supra. A representative method of expression in humancells is disclosed in Berg et al., BioTechniques 14(6):972-978 (1993).Suitable human cells include publicly available cell lines such as HeLAS3 (ATCC CCL2.2) and HEK293 (ATCC CRL1573). Expression in CHO cells isdisclosed, for example, in Asselbergs et al., Fibrinolysis 7:1-14(1993). Suitable hamster cell lines include CHO-K1(ATCC CCL61) and BHK(ATCC CRL6281), BHK-21 (ATCC 6281, CCL10 and CRL8544). Representativemonkey cells are CV-1 (ATCC CCL70), COS-7 (ATCC CRL1650), and VERO cells(ATCC CCL81). A suitable mouse cell line is C127 (ATCC 1804). Preferredcell lines are DHFR-minus CHO lines as disclosed in Uriaub et al., Proc.Natl. Acad. Sci. USA 77:4216-4220 (1980). Serum-independent cell linesare more preferred. See Kurano et al., Bio/Technology 16:245-258 (1990).In mammalian hosts, glycosylated or non-glycosylated forms of the C5aanalogues can be produced.

The C5a analogues isolated from transformed E. coli cells are renaturedto assume biological activity wherein the C-terminal cysteine is inreduced form, i,.e., it contains a free thiol group, preferably by usinga convenient one-step procedure. Applicants have unexpectedly discoveredthat treating the denatured C5a analogue with a redox couple in a molarratio of reducing agent to oxidizing agent from at least about 100:1 toabout 500:1 results in a biologically active C5a analogue having aC-terminal cysteine in reduced form. This ratio is from about 10-fold toabout 50-fold greater than known ratios (a preferred ratio of reducedsulfhydryl to oxidized sulfhydryl compound of 10:1 is disclosed on col.17, lines 43-45 of Builder et al., U.S. Pat. No. 4,620,948). Inaccordance with the procedure, the transformed E. coli cells, afterculturing under conditions sufficient to cause production of the C5aanalogue, are mixed with a denaturing and solubilizing agent, e.g., 6Mguanidine HCl, to produce denatured C5a analogue, optionally withfurther disruption by any known technique such as sonication, FrenchPress or DynoMill. The thus-mixed or thus-disrupted cells containing thedenatured C5a analogue are then mixed with a redox couple in a molarratio by weight of reducing agent/oxidizing agent of from at least about100:1 to about 500:1 under suitable conditions to produce renatured,biologically active C5a analogue. Suitable redox couples includecysteine/cystine and reduced glutathione/oxidized glutathione. Othersskilled in, the art will appreciate that other redox couples can beused. The glutathione redox couple is preferred. Suitable conditionsinclude a pH of from 6.5 to 7.5, preferably 7.4. The mixture is allowedto stand at room temperature for a time sufficient to maximize the yieldof protein. The preferred time is from about one-half hour to about 4hours. Thus, this method eliminates the need to isolate the refractile,inclusion bodies (i.e., the insoluble mass of expressed protein) fromthe bacterial cells, and then to reduce the thiol group of theC-terminal cysteine.

An embodiment where the C5a analogue or derivative thereof is expressedin the form of a fusion protein, the resultant inclusion body containingfusion protein can be recovered quantitatively after cell lysis usinglow speed centrifugation (e.g., about 3,000×g), thus providing arelatively highly enriched fraction which then can be appropriatelysolubilized. This procedure offers the advantage of using a 20-folddecrease in volume per weight of cells recovered from the fermenter.

In the alternative, the C5a analogues may be renatured according tostandard refolding and purification schemes such as disclosed in Builderet al., U.S. Pat. No. 4,620,948, for example. Following theseprocedures, the C-terminal cysteine will be in the form of an adduct,e.g., cys-cys or cys-glutathione. Therefore, the adduct must be furtherreduced to yield the free thiol group. Applicants have discovered thatadducts of the disclosed C5a analogues also function as C5a receptorantagonists which exhibit substantially no agonist activity as definedherein, and thus are included within the scope of the present invention.However, the further reduction would be necessary to prepare thepreferred embodiments if these standard renaturation techniques wereused.

Following renaturation, the C5a analogues may be purified to the extentdesired. Representative purification schemes include ultrafiltration,diafiltration, gel electrophoresis, chromatographic processes such asion exchange chromatography, size exclusion chromatography, HPLC,reverse phase HPLC, treatment with Sephadex, dialysis, affinitychromatography, etc. Those skilled in the art would appreciate that acombination of purification schemes can be used.

C5a analogues having a C-terminal cysteine residue can be oxidized toform dimers in accordance with standard techniques. To prepare thedimers, the thiol (--SH) groups of the C-terminal cysteines of therespective monomers (analogues) are oxidized to produce a disulfidelinkage. Homodimers and heterodimers are embraced by the term "dimer".

The instant C5a analogues and dimers thereof are useful in the treatmentand/or prevention of injurious conditions or diseases in which thecomplement system, and more particularly C5a and analphylatoxin, areinvolved. They are therapeutically most effective when administered toany mammalian patient, especially humans, who face a high risk ofC5a-mediated tissue destruction and death. In general, the conditions ordiseases are those such as inflammatory disorders where C5a is generatedin the serum proteolytically. Representative conditions responsive toC5a analogue therapy include pneumonitis, adult respiratory distresssyndrome (ARDS), idiopathic pulmonary fibrosis, pulmonary inflammationor injury, chronic progressive pulmonary dis-cystic fibrosis,byssinosis, asbestos-induced inflammation, myocardial infarction,post-myocardial infarction inflammation, ischemic heart damage, hepaticcirrhosis, primary biliary cirrhosis inflammation, chronic hepatitis,pancreatitis, hemorrhagic pancreatitis, inflammatory bowel disease,colitis, ischemic brain damage, encephalitis, cranial nerve damage inmeningitis, meningitis, uvetis, Purtscher's retinopathy, immunecomplex-mediated glomerulonephritis, renal cortical necrosis, gout,vasculitis, serum sickness, angio-edema, myasthenia gravis, systemiclupus erythematosis, rheumatoid arthritis, bullous skin disease,hypersensitivity, psoriasis, endotoxin shock, sepsis, severe trauma andburns. They can also be used therapeutically to treat patients sufferingfrom transplant rejection, those receiving immunosuppressive therapy ormassive blood transfusion, those exposed to medical devices, and thoseexperiencing pulmonary dysfunction following hemodialysis andleukopheresis.

The analogues and dimers thereof have further therapeutic utility asprophylactics, particularly in conditions caused by reperfusion, e.g.,reperfusion following ischemia, and circulatory contact with medicaldevices, as well as to prevent transplant rejection. In this case, theC5a analogue is administered suitably prior to or substantiallysimultaneously with the event that is known to cause the inflammation oraggravate an existing inflammatory condition.

The instant C5a analogues and dimers thereof can be administered by anytherapeutically effective route for a proteinacecous pharmaceutical,e.g., parenterally, intranasally, rectally or buccally, in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles as desired. The term"parenteral" embraces delivery modes such as subcutaneous, intravenous,intramuscular, instrasternal, intra-arterial injection and infusiontechniques.

Dosage amounts of the C5a analogues (and diners) of the presentinvention may be varied to achieve the desired therapeutic response fora particular patient. They will depend, for instance, on the activity ofthe particular antagonist, the mode of administration, the severity ofthe condition being treated, as well as the medical condition of thepatient. The determination of a therapeutically effective dosage amountfor a given condition and patient is within the level of skill in theart. In general, dosage levels of from about 1 ug to 100 mg per kilogramof body weight per day are administered daily to the mammalian host.Preferred dosage levels range from about 0.1 mg/kg to about 20 mg/kg ofbody weight per day. The C5a analogue is administered to the patient asa single continuous dose over a prolonged period of time. However, thetotal effective dosage may be divided into multiple doses, e.g., two tofour separate doses per day, if desired.

The C5a analogues (and dimers) of the present invention can beformulated into compositions using both known pharmaceuticallyacceptable ingredients and methods of preparation. See, e.g., Remingtonet al., Pharmaceutical Sciences, 15th Ed., Mack Pub. Easton (1975).Suitable compositions for parenteral administration comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, as well as sterile powders forreconstitution into sterile injectable solutions or dispersionsimmediately prior to use. Representative examples of suitable aqueousand nonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols, e.g., glycerol, propylene glycol, polyethylene glycol,and suitable mixtures thereof, vegetable oils, e.g., olive oil, andinjectable organic esters such as ethyl oleate. Fluidity may bemaintained by various Paeans including the use of coating materials suchas lecithin, the maintenance of required particle size (in the case ofdispersions) and surfactants.

The compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, dispersing agents, antibacterial andantifungal agents such as paraben, chlorobutanol, phenol and sorbic aid,isotonic agents such as sugars, sodium chloride, or agents which delayabsorption such as aluminum monostearate and gelatin. The C5a receptorantagonists may be incorporated into slow or sustained release ortargeted delivery systems such as polymer matrices, liposomes andmicrospheres.

Injectable formulations can be sterilized by numerous means, includingfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

Suspensions, in additon to the C5a analogue and any other activeingredient, may contain suspending agents such as ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth andmixtures thereof.

Compositions for rectal or vaginal administration are usually in theform of suppositories which can be prepared by mixing the polypeptidesof the present invention with suitable non-irritating excipients orcarriers such as cocoa butter, polyethylene glycol or a suppository waxwhich is solid at room temperature but liquid at body temperature, andtherefore melts in the rectum or vaginal cavity, and releases the C5areceptor antagonist.

Opthalmic formulations, eye ointments, powders and solutions are alsoincluded within the scope of the disclosed invention.

Polyclonal and monoclonal antibodies specific to the C5a analogues anddimers of the present invention may be prepared in accordance withstandard techniques. Polyclonal antibodies, for example, are raised byinjecting a C5a analogue-carrier protein conjugate into an animal, e.g.,rabbits, goats, sheep or horses, to raise anti-C5a analogue antibodies.See, e.g., A. Johnstone and R. Thorpe, Immunochemistry in Practice,Blackwell Scientific Publications, Oxford (1982). Monoclonal antibodiesspecific to the C5a analogues of the present invention may be preparedaccording to the techniques disclosed techniques disclosed in Kohler andMilstein, Nature 256:495-97 (1975). See also Peters, J. H., (eds.)Monoclonal Antibodies, Springer Verlag Berlin, Heidelberg, Germany(1992). The polyclonal and monoclonal antibodies specific to the C5aanalogues also exhibit substantially no cross-reactivity with human C5a.By the term "substantially no cross-reactivity," it is meant that: theanti-C5a analogue antibodies exhibit extremely low (negligible)cross-reactivity with human C5a such that no interference byendogenously produced C5a with the assay for the instant C5a analoguesin biological samples can be detected.

The C5a analogue-specific antibodies of the present invention areparticularly useful to detect: and quantify circulating C5a analogue ina subject previously administered with same, as well as in modulating,e.g., neutralizing, the activity of the circulating C5a analogue.Circulating C5a analogue can be detected in accordance with standardimmunological techniques which utilize antibodies. In general, a fluidor tissue sample is obtained from the subject and then reacted with anantibody specific to the C5a analogue which was administered to thesubject, under conditions suitable to allow for the detectable formationof an immune complex between the analogue and the antibody. Theformation of such an immune complex is indicative of the presence of theanalogue in the sample. The use of plasma or serum samples in suchassays are preferred. However, tissue such as certain blood cells, e.g.,PMNL's, can also be used. The presence and/or extent of reaction can bedetermined in a variety of methods known in the art such asradioimmunoassay, enzyme immunoassay, fluorescent immunoassay,fluorescent microscopy, etc., and the like. Qualitative and quantitativesuitable immunological assay methods are disclosed in J. Butler,Immunochemistry of Solid-Phase Immunoassay, CRC Press (1991)

Assays to detect circulating C5a analogue are typically employed tomonitor levels of the analogue during treatment. In addition, theantibodies of the present invention can be advantageously used in apharmaceutical composition to modulate or neutralizer the activity ofthe circulating C5a analog. The amount of antibody used will be a molarequivalent of the amount of analogue administered. The compositions maybe administered to a subject parenterally. Intravenous administration ispreferred especially in an emergency situation. The antibodies will beformulated in a unit dosage injectable form in association with apharmaceutically acceptable vehicle such as saline or Ringer's solution.

The invention will be further described by reference to the followingdetailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified.

EXAMPLE 1 Synthesis Of A Gene Encoding Human C5a

The human C5a gene was synthesized by oligonucleotide coupling. Thecodon usage of this synthetic gene was designed for optimal expressionin E. coli. The synthetic strategy is illustrated in FIG. 1. It entailedthe condensation of five fragments with the N-terminal residue changedfrom Thr to Met since the AUG codon gives a much higher frequency oftranslation initiation than any other codon. Fragment 1 encodes aShine-Delgarno sequence and the ATG start codon of the synthetic gene.Fragments 2-5 encode the C5a gene.

Oligonucleotide Synthesis:

Oligonucleotides were synthesized on a Gene Assembler (Pharmacia) by thesolid phase phosphoramidite method. The fully synthesizedoligonucleotides were cleaved from the solid support and deprotected byincubation with concentrated NH₄ OH for 16 h at 55° C. Theoligonucleotides were then purified by preparative gel electrophoresis.The acrylamide concentration used varied from 10% for oligonucleotidesgreater than 70 bases to 20% for those less than 40 bases in length.Following electrophoresis, the oligonucleotides were visualized by UVshadowing and. the major high molecular weight fragment was excised fromthe gel. The gel slice was pulverized in a test tube with a glass rodand the DNA extracted by incubation in 3.0 ml of 0.1 M triethylammoniunbicarbonate (TEAB) buffer at pH 7.5 for 16 h at 37° C.

The gel remnants were removed by centrifugation and the oligonucleotidesisolated by chromatography on SepPak C-18 columns (Waters Associates).The columns were pre-equilibrated by washing sequentially with 10 mlacetonitrile, 5 ml 30% acetonitrile in 50 mM TEAB and 10 ml 25 mM TEAB.The oligonucleotides were applied, washed with 10 ml 25 mM TEAB, andeluted from the columns with 5 ml 50% acetonitrile in 35.5 mM TEAB.Fractions were collected and those containing the oligonucleotides, asdetermined by absorbance at 260 nm, were dried in a SpeedVac (Savant).

Oligonucleotide Annealing and Coupling:

Prior to annealing, each oligonucleotide was phosphorylated at the5'end. The kinase reaction mixture contained 1 ug of oligonucleotide ina total volume of 40 ul, 77 mM TRIS, at pH 7.5, containing 12 mM MgCl₂,1 mM DTT (dithiothreitol) and 2 mM ATP. The reaction was initiated bythe addition of 10 units of T4 polynucleotide kinase and was allowed to5 proceed for 40 min at 37° C. 10 ul of sterile water were added to eachkination reaction and 48 ul of complimentary oligonucleotides wereadded, mixed and placed in a heating block at 78° C. The heating blockwas turned off and the mixture was allowed to cool to 30° C. The samplesthen were placed in a second heating block at 68° C. for 10 min, andagain the block was turned off and the mixture allowed to cool to 26° C.Annealed gene fragments were used to assemble the gene in phage M13mp18(New England Biolabs). The strategy for assembling the C5a encoding genein M13mp18 required three rounds of ligation reactions.

Following each ligation reaction of the appropriate gene fragments intoM13mp18, E. coli JM101 was transformed with the ligated M13 DNA.Isolation of the M13 phage from the recombinant clones was followed bysequence analysis of the construction. The final C5a gene was clonedinto M13mp18 to give M13mp18/C5a(Thr1Met, 1-74). The C5a(1-74) gene wassubsequently subcloned into a pB-6 vector, derived from plasmids pTZ19Rand pKK223-3 both obtained from Pharmacia) to yield pB-6/C5a(Thr1Met,1-74). See FIG. 2.

EXAMPLE 2 Site-directed Mutagenesis Of The C5a Gene

Using the oligonucleotide-directed in vitro Mutagenesis System Version 2(Amersham), the single stranded DNA from the C5a containing vectorM13mp18/C5a(1-74) and a mutagenic oligonucleotide, site-directedmutagenesis was performed. The mutation Cys27Ser in C5a was performedusing the mutagenic oligonucleotide, ACGGTGCTTCTGTTAACA (SEQ. ID. NO.6), following the procedure provided by the manufacturer. Four plaqueswere analyzed by dideoxy DNA sequencing for the correct Cys27Sermutation. Double stranded DNA was isolated from one of the correctmutant clones and restricted with PstI and BamHI. A 230 bp fragmentcontaining the mutation was subcloned into the pB-6 vector. Theresulting plasmid, pB-6/C5a(1-74, T1M, C27S), was sequenced again viathe dideoxy method to confirm the mutation.

EXAMPLE 3 Cassette Mutagenesis Of The C5a Gene

The plasmid pB-6/C5a(1-74, T1M, C27S) or pB-6/C5a (1-74, T1M,) wasrestricted with EcoRI and HindIII, and subcloned in the vector pWCB,also restricted with the same enzymes, to yield plasmids pWCB112,containing the gene encoding C5a(1-74, T1M, C27S), and pWCB100,containing the gene encoding for C5a(l-74, TLM), respectively. Then, theplasmids were used in cassette mutagenesis to make a series of newgenes. Oligonucleotides used in cassette mutagenesis were made with anApplied Biosystems 381A DNA Synthesizer, using solid phasephosphoramidite chemistry according to the instructions of themanufacturer. Ten ul TE, containing about 60 ug of pWC31112, were mixedwith 6 μl containing 60 U of PVUII and 3 μl containing 60 U of HindIII(New England Biolabs) and 10 μl of 10 ×High Salt buffer (1M NaCl, 0.5 MTris/HCl at pH 7.5, 0.1M MgCl₂, 10 mM DTT) and 71 μl ddH₂ O for a totalvolume of 100 ul. This solution was incubated at 37° C. for 16 hours.The thus-linearized vector of about 4.5 Kb was purified by preparativeelectrophoresis using a 1% agorose gel, followed by electroelution ofthe DNA fragment from the excised agarose gel slice. The recovered DNAfragment was transferred to an Eppendorf tube, 1 ml of absolute ethanolwas added and the tube centrifuged for 10 min at 14,000 rpm in anEppendorf centrifuge. The DNA pellet was dried under vacuum andsubsequently dissolved in 45 μl TE buffer (10 mM Tris.HCl at pH 7.4containing 1 mM EDTA) yielding pWCB112/A. single strandedoligonucleotides a 35 bp- sequence,5'CTGCGTGCTAACATCTCTCACAAAGACATGTGCTA3' (SEQ. ID. NO. 7), and a 39bp-sequence, 5 'AGCTTAGCACATGTCTTTGTGAGAGATGTTAGCACGCAG3 ' (SEQ. ID. NO.8), were purified by preparative electrophoresis on a 8% polyacrylamidegel. Following electrophoresis, the oligonucleotides were visualized byUV shadowing and the appropriate fragment excised from the gel. The gelslice was pulverized in a test tube with a glass rod and the DNAextracted by incubation in 3.0 ml of 0.1 M TEAB buffer at pH 7.5 for 16h at 37° C. The gel remnants were removed by centrifugation and theoligonucleotides isolated by chromatography on SepPak C-18 columns(Waters Associates). The columns were pre-equilibrated by washingsequentially with 10 ml acetonitrile, 5 ml 30% acetonitrile in 50 mMTEAB and 10 ml 25 mM TEAB. The oligonucleotides were applied, washedwith 10 ml 25 mhM TEAB, and eluted from the columns with 5 ml 50%acetonitrile in 35.5 mM TEAB.

Fractions were collected and those containing the oligonucleotides, asdetermined by absorbance at 260 nm, were dried in a SpeedVac (Savant).Annealing of the 35 and 39 bp oligonucleotides to form double strandedDNA for ligation into the restricted vector pWCB112/A was performed bymixing equal amounts of each oligonucleotide with Klenow Buffer, whichcontains 0.05 M Tris/HCl at pH 7.6 containing 0.01 M MgCl₂, heating thesample to 95° C. for 10 min and subsequent cooling to room temperatureover a 2 hour period. The double-stranded DNA was ligated into therestricted vector pWCB112 using a 3-fold excess of insert over vectorwith 1 μl, 2 U, of T4 DNA ligase (BRL). The reaction was run for 17hours at 4° C.

Using essentially the same technique, a number a molecules were preparedmerely by using different oligonucleotides, and either pWCB100 orpWCB112. They are set forth below in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    C5a analogues produced by cassette mutagenesis of the C5a(1-74) or            C5a(1-74,C27S) gene                                                           Analogue No.                                                                         oligonucleotide sequences used and encoded C5a analogue                __________________________________________________________________________    1.     C5a(1-64,T1M,C27S,N64C)                                                       CTGCGTGCTTGCTA (SEQ. ID. NO. 9)                                               AGCTTAGCAAGCACGCAG (SEQ. ID. NO. 10)                                   2.     C5a(1-65,T1M,C27S,I65C)                                                       CTGCGTGCTAACTGCTA (SEQ. ID. NO. 11)                                           AGTTAGCAGTTAGCACGCAG (SEQ. ID. NO. 12)                                 3.     C5a(1-66,T1M,C27S,S66C)                                                       CTGCGTGCTAACATCTGCTA (SEQ. ID. NO. 13)                                        AGCTTAGCAGATGTTAGCACGCAG (SEQ. ID. NO. 14)                             4.     C5a(1-67,T1M,C27S,H67C)                                                       CTGCGTGCTAACATCTCTTGCTA (SEQ. ID. NO. 15)                                     AGCTTAGCAAGAGATGTTAGCACGCAG (SEQ. ID. NO. 16)                          5.     C5a(1-68,T1M,C27S,K68C)                                                       CTGCGTGCTAACATCTCTCACTGCTA (SEQ. ID. NO. 17)                                  AGCTTAGCAGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 18)                       6.     C5a(1-69,T1M,C27S,D69C)                                                       CTGCGTGCTAACATCTCTCACAAATGCTA (SEQ. ID. NO. 19)                               AGCTTAGCATTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 20)                    7.     C5a(1-70,T1M,C27S,M70C)                                                       CTGCGTGCTAACATCTCTCACAAAGACTGCTA (SEQ. ID. NO. 21)                            AGCTTAGCAGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 22)                 8.     C5a(1-71,T1M,C27S,Q71C)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGTGCTA (SEQ. ID. NO. 23)                         AGCTTAGCACATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 24)              9.     C5a(1-72,T1M,C27S,L72C)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGCAATGCTA (SEQ. ID. NO. 25)                      AGCTTAGCATTGCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 26)           10.    C5a(1-73,T1M,C27S,G73C)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGCAACTGTGCTAS (SEQ. ID. NO. 27)                  AGCTTAGCACAGTTGCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 28)        11.    C5a(1-74,T1M,C27S,Q71C)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGTGCCTGGGTCGTTA (SEQ. ID. NO. 29)                AGCTTAACGACCCAGGCACATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO.                30)                                                                    12.    C5a(1-73,T1M,C27S,Q71C)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGTGCCTGGGTTA (SEQ. ID. NO. 31)                   AGCTTAACCCAGGCACATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 32)        13.    C5a(1-72,T1M,C27S,Q71C)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGTGCCTGTA (SEQ. ID. NO. 33)                      AGCTTACAGGCACATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 34)           14.    C5a(1-74,T1M,R74C)                                                            CTGCGTGCTAACATCTCTCACAAAGACATGCAACTGGGTTGCTA (SEQ. ID. NO. 35)                AGCTTAGCAACCCAGTTGCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO.                36)                                                                    15.    C5a(1-71,T1M,C27S)                                                            CTGCGTGCTAACATCTCTCACAAAGACATGCAATA (SEQ. ID. NO. 37)                         AGCTTATTGCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 38)              16.    C5a(1-71,T1M,C27S,Q71D)                                                       CTGCGTCCTAACATCTCTCACAAAGACATGGACTA (SEQ. ID. NO. 39)                         AGCTTAGTCCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 40)              17.    C5a(1-71,T1M,C27S,Q71S)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGTCTTA (SEQ. ID. NO. 41)                         AGCTTAAGACATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 42)              18.    C5a(1-71,T1M,C27S,Q71H)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGCACTA (SEQ. ID. NO. 43)                         AGCTTAGTGCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 44)              19.    C5a(1-71,T1M,C27S,Q71R)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGCGTTA (SEQ. ID. NO. 45)                         AGCTTAACGCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 46)              20.    C5a(1-71,T1M,C27S,Q71L)                                                       CTGCGTGCTAACATCTCTCACAAAGACATGCTGTA (SEQ. ID. NO. 47)                         AGCTTACAGCATGTCTTTGTGAGAGATGTTAGCACGCAG (SEQ. ID. NO. 48)              21.    C5a(1-71,T1M,C27S,H67F,Q71C)                                                  CTGCGTGCTAACATCTCTTTCAAAGACATGTGCTA (SEQ. ID. NO. 49)                         AGCTTAGCACATGTCTTTGAAAGAGATGTTAGCACGCAG (SEQ. ID. NO.                  __________________________________________________________________________           50)                                                                

Analogue Nos. 10-20 are agonists, and are outside the scope of thepresent invention. They are included for purposes of comparison. Thepreparation of the dimeric form of analogue No. 8 is described inExample 5c, below. It is designated Analogue No. 22.

The complete nucleotide sequence of the polynucleotide encoding C5aanalogue No. 21 is set forth in Table 3 below.

                                      TABLE 3                                     __________________________________________________________________________     ##STR8##                                                                      ##STR9##                                                                     GCTAAGTACAAACACTCTGTTGTTAAAAAA                                                 ##STR10##                                                                    GACGAAACTTGCGAACAGCGTGCTGCTCGT                                                 ##STR11##                                                                     ##STR12##                                                                    CTGCGTGCTAACATCTCTTTCAAAGACATG                                                 ##STR13##                                                                    __________________________________________________________________________

However, those skilled in the art will appreciate that manypolynucleotides can be prepared encoding the identical C5a analogue, dueto the degeneracy of the genetic code. See, e.g., Watson et al.,Recombinant DNA, 2nd Ed., Freeman, N.Y. (1993).

EXAMPLE 4 Expression Of C5A And C5a Analogues In E.coli

To achieve expression, the synthetic genes for the C5a analogues setforth in Table 1 were subcloned in the pWCB vector, the modifiedexpression vector pKK223-3 (Pharmacia) having a BamHI site deleted and aPvuII site changed to a PvuI site, and containing theisopropyl-thio-beta-D-galactoside (IPTG)-inducible tac-promotor, and anampicillin resistance gene. E. coil strain LCIQ, is a derivative ofstrain LC137 (lon, htpR) disclosed in Goff et al., Proc. Natl. Acad. SciUSA 81:6647-6651 (1984). It contains an F' factor encoding lacIQ, fromstrain DH5alpha F'IQ (BRL laboratories), and was the host for theexpression plasmids. E. coli LCIQ containing the appropriate expressionplasmid was grown at 30° C. in LB broth until an OD₅₅₀ of 1 was reached.The culture was induced for 3 hours with IPTG at 2.5 mM finalconcentration. The cells were harvested by centrifugation and stored at-80° C. until use.

EXAMPLE 5a Refolding And Purification Of C5a And C5a Analogues

Recombinant protein was isolated from the frozen E. coli cell pastealiquots from Example 4 after thawing in a buffer containing 6 Mguanidinium hydrochloride (5:1 v:w, buffer:cell paste). The cells werethen disrupted by sonication, and the product dialyzed overnight againsta 50 mM Tris/HCl buffer at pH 8.0 containing either 1 mM cysteine and 1mM cystine or 1 mM reduced/oxidized glutathione to promote renaturation.The dialysate; was then acidified to pH 3 by the addition of 6 N HCl.The precipitate was removed by centrifugation and the supernatant waspurified on a DeltaPak C18,100 Å, 15 micron, reverse phase HPLC column(Waters) using a linear gradient from 25% to 35% acetonitrile in waterin the presence of 0.1% TFA over 30 min. The major peak eluting from thecolumn at about 28% acetonitrile was collected and lyophilyzed. Thisfraction contained the gluthathione adduct of recombinant C5a analogue(adducts of analogues 5, 7, 8, 9, and 21 in Table 1), or thecysteine-adduct of the C5a analogue (the adduct of analogue No. 8, Table1).

Approximately 0.002 mmoles of the cysteine-adduct of the C5a analoguegene product were dissolved in 50 ml 0.1 M Tris buffer at pH 7.4. 0.02mmoles of DTT were added. After 4 hours, about 80% of the C-terminalcys-cys linkages was converted to the free cysteine and the product waspurified on a C4, 15 micron, 300 Å, reverse phase column (Alltech) usinga linear gradient from 25 to 35% acetonitrile in water in the presenceof 0.1% TFA for 30 min. Fractions containing the product eluting atabout 29% acetonitrile were lyophilized and then stored at 4° C.,dessicated under vacuum.

EXAMPLE 5b Refolding And Purification Of C5a And C5a Analogue

Recombinant protein was isolated from the frozen cell E. coli pastealiquots from Example 4 after thawing in a buffer containing 6Mguanidinium hydrochloride (5:1 v:w, buffer:cell paste). The cells werethen disrupted by sonication and the product was diluted twenty-foldwith 100 mM Tris/HCl buffer at pH 7.4 containing 1. mM reduced/0.01 mMoxidized glutathione. After 4 h, the solution was acidified to pH 3 bythe addition of 6 N HCl. The resulting precipitate was removed bycentrifugation and the supernatant was purified on a DeltaPak C18, 100Å, 15 micron, reverse phase HPLC column (Waters) using a linear gradientfrom 25 to 35% acetonitrile in the presence of 0.1% TFA over 30 min. Themajor peak eluting from the column at about 30% acetonitrile wascollected and lyophilized. The thus-isolated C5a analogue had aC-terminal cysteine having a reduced thiol group.

EXAMPLE 5c Formation Of C5a Analogue Dimers

C5a analogues having in their C-terminal region a free thiol group wereconverted from the monomeric form (after refolding as described inExample 5b) to a dimeric form.

The recombinant protein was isolated from the frozen E. coli pastealiquots from Example 4 after refolding according to Example 5b using a1 mM reduced/0.01 mM oxidized glutathione mixture in 100 mM Tris/HCl atpH 7.4. After 4 h, the solution was acidified to pH 3 by the addition of6 N HCl. The resulting precipitate was removed by centrifugation and thesupernatant absorbed on a SP-Spherodex\ ion exchange column,equilibrated with 25 mM buffer at pH 7.0. After washing the column with25 mM Tris at pH 7.0, the C5a analogue was eluted from the column with25 mM Tris at. pH 7.0, containing 0.75 M NaCl. The partially purifiedC5a analogue was brought to pH 3.0 with formic acid, and diluted withdistilled water to achieve a protein solution having a conductivity ofabout 45 mS/cm, and absorbed to a SP-High Performance ion exchangecolumn equilibrated in 50 mM formic acid at pH 3.5, containing 0.6 MNaCl. C5a analogue was eluted from the column using a linear gradientfrom 0.6-1.0 M NaCl in 50 mM formic acid buffer at pH 3.5.

The major peak eluting from the column at about 0.725 M NaCl wascollected. The thus-isolated C5a analogue had a C-terminal cysteinehaving a reduced thiol group. Adjustment of the pH to 7.0 with a 25%aqueous ammonia solution and storage of the solution resulted in aconversion of the molecule to its dimeric form. At pH 7.0 and a proteinconcentration of about 0.3-0.6 mg/ml and storage at 4°8° C., theconversion was at least 80% completed in 2 days. The dimeric form of theC5a analogue was finally purified on a DeltaPak C18, 100 Å, 15 micron,reverse phase HPLC column (Waters) using a linear gradient from 25% to40% acetonitrile in the presence of 0.1,) TFA over 30 min. The majorpeak eluting from the column at about 33% acetonitrile was collected andlyophilized. The thus-isolated molecule was a dimer of the C5a analogueproduced by the E. coli expression system.

EXAMPLE 6 Receptor Binding Assay

C5a and C5a receptor antagonists were tested for their affinity for theC5a receptor. Binding of 125I! Bolton-Hunter (BH)-labelled C5a, preparedas described in Harris et al., J. Receptor Res. 11:115-128 (1991), toPMNL membranes was measured as described in Rollins et al., J. Biol.Chem. 263:520-526 (1988), with modifications as described in Braunwalderet al., Mol. Immunol. 29(11):1319-1324 (1992). PMNLs were resuspended inHanks balanced salt solution, without Ca⁺⁺ and Mg⁺⁺ and which contained10 mM HEPES; at pH 7.3, 2.5 mM MgCl₂, 100 units/ml DNAse I, 0.1 mMPMISF, 10 μg/ml aprotonin and 10 μg/ml leupeptin. They were thenequilibrated at 400 psi for 20 min at 4° C. in a nitrogen cavitationbomb. After evacuation into 3 volumes 0.5 M KHCO₃ containing 25 mM EDTAand the protease inhibitors listed above, the gelatinous material wasremoved with forceps and the mixture was centrifuged at 400×g for 10 minat 4° C. The resulting supernatant was centrifuged at 50,000×g for 60min at 4° C. The pellets from the aliquots representing 200×10⁶ cellswere stored at -70° C. For binding studies, these membranes wereresuspended at an equivalent of 20×10⁶ cells/ml in 50 mM HEPES at pH7.3, containing 1 mM CaCl₂, 5 mM MgCl₂, 0.1 mM PMSF, 0.1% bacitracin and0.5% BSA. After further 1:75 dilution with the same buffer, 400 ul ofthis suspension were added to duplicate tubes containing 50 ul of125I!BH-C5a (specific activity 2200 Ci/mmol, final concentration 4.0pM), and 50 ul buffer or C5a analogues to be tested at variousconcentrations for inhibitor properties.

Nonspecific binding was determined in the presence of 10 nM unlabelledC5a. The binding reaction was initiated by the addition of the PMNLmembranes and was continued for 120 min at 4° C. Bound and freeradioactivity were separated by vacuum filtration through GF/C glassfiber filters (Whatman), pretreated for 90 min with 0.05% PEI(polyethyleneimine) using a Cell Harvester (Brandel, Gaithersburg, Md.).Filters were washed with 3×5 ml of ice-cold 5 mM Tris buffer at pH 7.4and counted in a multiwell Gamma counter (Genesys). Data were analyzedusing the non-linear regression analysis program, RS/1 (Bolt, Beranekand Newman, Boston) and expressed as IC₅₀ values. The results are setforth below in Table 4 as K_(i) values using the Cheng-Presoff equation.See Braunwalder et al., supra

                  TABLE 4                                                         ______________________________________                                        Receptor binding studies of C5a analogues                                                         Receptor Binding                                          C5a analogue        Ki.sup.(nM)                                               ______________________________________                                        9 Glutathione       1.85                                                      8 Glutathione       1.7                                                       7 Glutathione       7.2                                                       8 Cys               2.8                                                       9                   0.9                                                       8                   0.2                                                       7                   0.4                                                       15*                 3.0                                                       16*                 8.5                                                       17*                 7.5                                                       18*                 0.15                                                      19*                 0.35                                                      20*                 0.035                                                     21                  0.1                                                       22                  0.04                                                      C5a                 0.0035                                                    ______________________________________                                         *= agonist; numbers in left column refer to Table 1.                     

These results demonstrate that the C5a analogues of the presentinvention competitively displace wild-type C5a with nanomolar K_(i) s.

The C5a analogues of the present invention have an affinity for the C5areceptor measured as a K_(i) in the competitive displacement assaydisclosed in Braunwalder et al., supra. (using the radioligand,125I!Bolton-Hunter labelled C5a), of less than about 1.0×10⁻⁸ M,preferably less than about 2.0×10⁻⁹ M, and more preferably less thanabout 1.0×10⁻¹⁰ M.

EXAMPLE 7 C5A Induced Ca++ Rise

Recombinant human C5a was dissolved in Hanks buffer containing 0.01%Tween-20, and all stock dilutions of C5a were made in this buffer. Theacetoxymethyl ester of fura-2 (fura 2AM, Molecular Probes) was dissolvedin DMSO. Neutrophils were purified from human peripheral blood bysedimentation in 6% hetastarch (HESPAN, DuPont, Waukegan, Ill.),followed by counter flow elutriation as described in Chapman-Kirkland etal., J. Immunol. Meth. 142:95-104 (1991). Purified cells (2×10⁶ /ml)were mixed with 0.2 uM fura- 2AM and incubated for 30 min at 37° C. inHEPES buffered Hanks solution without calcium or magnesium. Fifteenminutes before the assay, the cell suspension was transferred to acurvette containing a stir bar and calcium was added to 1 mM. The cellsuspension was incubated with stirring at 37° C. Assays were terminatedwithin 5 h of cell purification and a standard control response wasobtained periodically to insure that the cell responses were notchanging over the time of the experiment. The amount of fluorescence wasdetermined using an SLM 8000 spectrofluorometer (SLM-Aminco Instruments,Urbana, Ill.). Curvettes were placed in the fluorometer and afterobtaining a baseline for 10 sec, the C5a receptor antagonists to betested for antagonistic properties were added and any change influoresence excitation ratio of 340 nm/380 nm (emission of 510 nm) wasmeasured. Forty seconds after analogue addition, a challenge dose of C5awas added to a final concentration of 100 pM and the resulting change inexcitation ratio was measured.

IC₅₀ values were used as a measure of antagonist potency. These valuesare defined as the concentration of C5a analogue needed to reduce thecalcium rise response of the 100 pM C5a challenge dose by 50%. EC₅₀values were used as a measure of agonist potency. EC₅₀ is defined asthat concentration of C5a analogue that elicited 50% of the maximumcalcium rise response produced by the analogue. The results are setforth below in Table 5.

                  TABLE 5                                                         ______________________________________                                        C5a induced calcium rise studies on C5a analogues                                        Calcium Rise (nm)                                                               IC.sub.50    EC.sub.50                                           analogue     (antagonist) (agonist)                                           ______________________________________                                        9 glutathione                                                                              1000         NM(not measurable)                                  8 glutathione                                                                              2000         NM                                                  7 glutathione                                                                              2000         NM                                                  8 cysteine   105          NM                                                  9            43           NM                                                  8            14           NM                                                  7            54           NM                                                  15           NM           90                                                  16           NM           310                                                 17           NM           120                                                 18           NM           150                                                 19           NM           40                                                  20           NM           75                                                  21           6            NM                                                  22           10           NM                                                  C5a          NM           0.07                                                ______________________________________                                    

Ca5 analogue No. 7 was tested up to a concentration of 1.0×10⁶ M. C5aanalogue No. 8 was tested up to a concentration of 3.0×10⁻⁶ M, analogueNo. 9 was tested up to a concentration of 1.5×10⁶ M, and analogue No. 21was tested up to a concentration of 8.0×10⁻⁷ M. Agonist activity was notdetected in all cases. The results, analyzed collectively with those setforth in Table 2, above, suggest that the analogues of the presentinvention function as competitive inhibitors of C5a. They demonstratethat the C-terminus of the analogues should be an uncompleted cysteineor a cysteine residue which is complexed through a disulfide linkagewith another C5a analogue of the present invention, to achieve thehighest potency.

EXAMPLE 8 Rabbit Dermal Model Of Inflammation

All experiments were performed on male New Zealand White rabbitsweighing 2.5-3.0 kg. The backs; of the rabbits were shaved and 40-50skin sites were designated with markers of different colors. Differentstimuli (i.e., C5a, C5a analogue, C5a+C5a analogue, vehicle control,etc.) were injected intradermally at 0.1 ml/site using a sterile,disposable, 26 gauge, 0.5 in. needle and 1.0 cc tuberculin syringe. I.D.injections were administered in replicates of six, roughly 45 minutesbefore euthanization. C5a alone was injected at a dose of 50 ng/site,and the C5a receptor antagonists were co-injected at variousconcentrations with the same dose of C5a. At 20 minutes prior toeuthanization, 18-36 uCi of 125I!-labeled bovine serum albumin in 1.0 mlphysiological saline were introduced into the systemic circulation viathe marginal auricular vein. At 45 minutes, the rabbit was euthanizedwith an I.V. overdose of sodium pentobarbital. A 5.0 ml sample ofperipheral blood was secured via cardiac puncture, centrifuged at 2000rpm for 10 minutes, and 1.0 ml of plasma was collected and used as areference to determine the amount of 125I in the plasma.

After death, the dorsal skin was excised and pinned to a woodendissecting board. Blood in the major vasculature of the skin wasmanually expressed toward the periphery. This procedure reducedvariation among skin sites and decreased background radioactivity.Inflammatory lesions were then punched out of the skin with the aid of a15 mm cork borer and mallet and deposited in 12×75 mm polystyrene tubes.Injection sites were then analyzed for their radioactive content using aGamma Counter (Genesys). The amount of 125I!-bovine serum albumin (BSA)that exuded from the blood vessels and which was localized at theinflammatory sites was found to be directly proportional to the degreeof enhancement in vascular permeability. The ID₅₀ value of the C5aanalogue is the dose of that C5a analogue causing a 50% reduction in theradioactivity produced by 50 ng C5a co-injected at the same site.

C5a analogue No. 8 (in Table 1) was found to possess an ID₅₀ of 70ng/site, and did not cause a pro- inflammatory reaction at the dose of175 ng/site. This result demonstrates that the analogue is anantagonist: in vivo and does not exhibit agonist properties in vivo.

EXAMPLE 9 C5a-Induced Neutropenia In The Rabbit

All experiments were performed on male New Zealand White rabbitsweighing 2.5-3.0 kg. Rabbits were anesthetized with 10 mg/kg xylazineand 50 mg/kg ketamine administered in combination intramuscularly. A 25gauge butterfly catheter was inserted in the lateral ear vein to use forinfusions. Each blood sample (0.2 ml) was collected from the central earartery into a plastic syringe fitted with a 25 gauge, 5/8 inch needleand charged with 7.5% EDTA as an anticoagulant. Blood was immediatelyexpressed into a microcentrifuge tubes containing 10 microliters of 7.5%EDTA. An initial arterial blood sample (#1) was obtained and immediatelythereafter vehicle or the C5a analogue of Example 8 was infusedintravenously (bolus injection). Twenty seconds later, a second bloodsample (#2) was obtained and twenty seconds thereafter 100 ng of C5a in0.2 ml were infused intravenously (bolus injection). Twenty secondslater, a third blood (#3) sample was taken. Thirty minutes later, asecond round of blood sample (#4)--20 seconds--C5a infusion--20seconds--blood sample (#5) was performed. Blood samples were evaluatedby automated hematologic analysis (Technicon H*1) using softwarespecific for rabbit blood. Reductions in neutrophil counts (number permilliliter) induced by C5a(C5a-induced neutropenia, determined bycomparing blood sample #3 to #2 and #5 to #4) were compared betweenvehicle-treated and C5a analogue-treated animals. The C5a analogue didnot alter baseline neutrophil counts from normal; i.e., the C5a analoguedid not exhibit agonistic (C5a-like) properties.

C5a-induced neutropenia in the C5a analogue-treated rabbits wassignificantly (P>0.05) inhibited as compared to vehicle-treated rabbitsby 67% and 41% at the 40-second and 30-minute C5a challenge intervals,respectively. These results demonstrate the efficacy of administeringthe C5a analogues systemically.

EXAMPLE 10 Comparative Receptor Binding And C5a Induced Calcium Rise OfC5a Analogues with the DecapeptideH-Ile-Ser-Phe-Lys-Asp-Met-Gln-Leu-Gly-Arg-OH (SEQ. ID. NO. 52).

Five C5a analogues (Nos. 5, 7, 8, 9 and 10 from Table 2) were preparedand run in C5a receptor binding and C5a receptor calcium rise assays andcompared with the synthetic decapeptide disclosed in Table I (No. 14) inOr et al., J. Med. Chem. 35:402-406 (1992). Results of this experimentare shown in Table 6 below.

                  TABLE 6                                                         ______________________________________                                                 Receptor Binding                                                                          Ca.sup.++  Rise                                          Compound   Ki (nM)       IC.sub.50 (nM)                                                                          EC.sub.50 (nM)                             ______________________________________                                        Analogue 5 0.06          84                                                   Analogue 7 0.35          859                                                  Analogue 8 0.04          51                                                   Analogue 9 0.15          621                                                  Analogue 10                                                                              0.03                    0.6                                        C5a(1-74)  0.0035                  0.07                                       Decapeptide                                                                              5,000                   2058                                       ______________________________________                                    

These results demonstrate that the C5a analogues of the presentinvention tested possess a 14,000-125,000 fold greater binding affinityfor the receptor than the decapeptide. The above data also show that theC5a analogues of the present invention are C5a receptor antagonistsmolecules that exhibit substantially no agonist activity, while thedecapeptide exhibits significant agonist activity.

EXAMPLE 11 Preparation Of Polyclonal Antibodies Specific ToC5a(1-71,T1M,C27S,Q71C)

Antigen preparation

1 mg of C5a(1-71,T1M,C27S,Q71C) was conjugated to 2 mg Keyhole Limpethemocyanin (KLH) using the Imject\Immunogen EDC conjugation kit fromPierce Chemical Co. (Rockford, Ill., USA), following the manufacturer'sdirections. Conjugation efficiency was followed by adding 3,500 cpm of¹²⁵ I-C5a (New England Nuclear, Boston, Mass.). The final volume of theconjugate was 2.25 ml containing 0.34 mg C5a(1-71,T1M,C27S,Q71C) (0.15mg/ml) and an estimated 0.9 mg/ml of KLH.

Production of anti-C5a(1-71,T1M,C27S,Q71C) antiserum

C5a(1-71,T1M,C27S,Q71C) conjugate (0.5 ml) was homogenized with 0.5 mlof Freund's Complete Adjuvant (Sigma Chemical Co., St. Louis, Mo).Female New Zealand White rabbits, purchased from Millbrook Farms(Amherst, Mass.), were injected subcutaneously in two sites (0.2 mlhomogenate per site) in the scapular areas. After 21 days the procedurewas repeated. Further injections were carried out using Freund'sIncomplete Adjuvant (Sigma); the third injection was given after a totalof 55 days, and a fourth at 126 days. Blood (ca. 30 ml) was taken fromthe rabbits between 3 and 5 weeks after each injection, allowed to clotand the serum removed.

Peptide immobilization for antibody adsorption

C5a(1-71,T1M,C27S,Q71C) or C5a (1 mg) was conjugated to 2 mg Bovineserum albumin (BSA), using the Imject\Immunogen EDC conjugation kit fromPierce Chemical Co. and ¹²⁵ I-C5a to follow efficiency, as describedabove. The final volume of C5a(1-71,T1M,C27S,Q71C)/BSA was 2.25 ml at0.25 mg/ml C5a(1-71,T1M,C27S,Q71C); for C5a/BSA the final volume was2.25 ml at 0.32 mg/ml C5a. Both conjugates contained an estimated 0.9mg/ml BSA.

The two peptide conjugates were dialysed against 0.2M sodium hydrogencarbonate buffered to pH 8.6 with sodium carbonate. For each conjugate,2 ml of AH(aminohexyl)-Agarose gel (Sigma Chemical Co., St. Louis, Mo.)prewashed in the same buffer was activated by adding gluteraldehyde to afinal concentration of 1% v/v and incubating for 15 min at 20° C. Thegel was washed thoroughly in buffer to remove gluteraldehyde, then theconjugate solutions were added and incubated at 20° C. for 1 hr. Theuncoupled protein was rinsed away from the gel and remaining bindingsites were blocked by overnight incubation at 4° C. with 20 ml 0.2Mglycylglycine. The gel was packed into a 0.5 cm×10 cm glass column andwashed thoroughly with Dulbecco's phosphate-buffered saline pH 7.2containing 0.1% sodium azide (PBS-A).

Affinity chromatography

Serum from rabbits immunized with C5a(1-71,T1M, C27S,Q71C) was passedthrough the C5a/BSA column at 2 ml/hr. The absorbed antiserum emergingfrom the column was collected. The column was washed thoroughly with0.5M NaCl buffered with 0.05M sodium phosphate to pH 7.2 and containing0.1% sodium azide. Bound antibody was removed with 3M ammoniumthiocyanate. The eluting antibody was detected using an in-line UVmonitor reading at 280nm and set at 0.2 OD maximum deflection. On thefirst two passages of serum the eluting antibody was collected andimmediately dialysed against PBS-A, then concentrated by ultrafiltrationto around 1 mg/ml. This process was repeated several times for eachserum batch. Sera were considered to be absorbed when no more proteinwas detected eluting from the C5a column.

The absorbed antiserum was then passed through theC5a(1-71,T1M,C27S,Q71C) immuno-absorbent column. Bound antibody waseluted with 3M ammonium thiocyanate and immediately dialysed againstPBS-A, then concentrated to ca. 1 mg/ml.

EXAMPLE 12a Preparation Of Labelled Antibody To Detect boundC5a(1-71.T1M,C27S,Q71C)

The anti-C5a(1-71,T1M,C27S,Q71C) antibody eluted from the C5a column(i.e., antibody which cross-reacts with C5a) was conjugated to alkalinephosphatase: 1.4 it mg of antibody in 1 ml of PBS was added to 5 mg(5,000 units) of alkaline phosphatase (Type VII-T, Sigma Chemical Co.).Gluteraldehyde was added to a final concentration of (0.2% v/v. The mixwas incubated at 20° C. for 90 min, then dialysed overnight againstPBS-A at 4° C. The buffer was changed to 0.05M Tris buffer, pH 8.0containing 1 mM magnesium chloride, and dialysed overnight at 4° C.

EXAMPLE 12b Detection Of Bound C5a(1-71,T1M,C27S,Q71C) Via EILISA

Specifically purified rabbit anti-C5a(1-71,T1M,C27S,Q71C) at 0.57 mg/mlwas diluted 1:500 in 0.1M sodium borate/boric acid, pH 8.6. ELISA plates(Maxisorp\, Nunc, Naperville, Ill.) were coated with 100 ul/well of thissolution for 4hr at 20° C. The plates were washed three times to removeunbound material. Samples containing C5a(1-71,T1M,C27S,Q71C) or standardpreparations of C5a(1-71,T1M,C27S,Q71C), suitably diluted PBS-A+1% BSA(PBS/BSA), were added to the wells in 100 μl, for 4 hr at 20° C.Labelled antibody was added at 1:3000 in 100 μl PBS/BSA and incubatedovernight at 4° C. The plates were washed and then enzyme substrate (foralkaline phosphatase, p-nitrophenyl phosphate (Sigma Chemical Co.) at 1mg/ml in 10% v/v diethylamine pH 9.8) was added. Color development wasallowed to proceed at 20° C. in the dark for about 5 hr. The plates wereread at 405 nm using a Biomek 1000 (Beckman Instruments, Calif., USA).

Using the same conditions described above, a standard curve ofC5a(1-71,T1M,C27S,Q71C) was constructed (data not shown).

EXAMPLE 12c Specificity Of Affinity Purified SpecificAnti-C5a(1-71,T1M,C27S,Q71C)

C5a(1-71,T1M,C27S,Q71C) or C5a was used to coat microtiter plates at 1μg/well in 100 ul coating buffer for 4 hr at 20° C. The plates werewashed and serial dilutions of the antibody eluted from C5a(1-71, T1M,C27S, Q71C) after absorption on C5a were made into the plate wells in100 μl PBS/BSA. After 4 hr incubation at 20° C., the plates were washedagain. Binding of rabbit antibody was detected with goatanti-rabbit/horseradish peroxidase (Pierce Chemical Co.) at 1:1000 inPBS/BSA, 100 μl/well. After incubating with the second antibody for 4 hrat 20° C., the plates were washed and horseradish peroxidase activitydemonstrated with 2,2'-azinobis(3-ethylbenzothiazoline)-6 sulfonic aciddiammonium salt ABTS substrate (Pierce Chemical Co.). After 30 mindevelopment, the color was read at 405 nm.

EXAMPLE 13 Measurement Of C5a(1-71.T1M,C27S,Q71C) In Rabbit PlasmaSamples

Blood was sampled into heparin coated tubes from two anesthetizedrabbits (#1 and #2) which were then injected intravenously withC5a(1-71,T1M,C27S,Q71C). Blood was collected after a further 30 mininterval. A further injection of C5a(1-71,T1M,C27S,Q71C) was then givenand blood was again collected after 30 min. This was repeated 4 moretimes. The samples were centrifuged to remove blood cells, and theplasma was removed and stored at -20° C. until used in the ELISA.

The samples were diluted in PBS/BSA and quantified in the ELISA againstthe standard curve generated in Example 11. The increase in circulatingC5a(1-71,T1M,C27S,Q71C) with time was then determined. No activity couldbe detected in samples taken from the two rabbits before injection ofC5a(1-71,T1M,C27S,Q71C), demonstrating the specificity of the antibody.The results also demonstrate that the antibody exhibits nocross-reactivity with rabbit C5a, and that C5a(1-71,T1M,C27S,Q71C) isnot a naturally occurring substance in rabbits.

EXAMPLE 14 Comparison Of C5a(1-71,T1M,C27S,Q71C) In Rabbit CirculationWith Standard C5a(1-71,T1M,C27S,Q71C): Use Of The SpecificAnti-C5a(1-71,T1M,C27S,Q71C) Antibody As An Antidote ForC5a(1-71,T1M,C27S,Q71C)

The plasma sample obtained from the last time point of rabbit #2 (fromExample 13) was subjected to serial doubling dilutions and the slope ofthe curve obtained was compared with the slope of the standard curve.The two slopes were parallel, indicating that C5a(1-71,T1M,C27S,Q71C)which had been circulating in the rabbit still retained its antigenicproperties and would be recognized in the ELISA in the same manner asthe standard C5a(1-71,T1M,C27S,Q71C). This result also indicates thatthe analogue would be neutralized by this antibody if removal of theC5a(1-71,T1M,C27S,Q71C) from the circulation should become necessary.

EXAMPLE 15 Preparation Of Monoclonal Anti-C5a(1-71 T1M,C27S,Q71C)

Preparation of monoclonal antibodies was carried out in BALB/c miceusing standard procedures developed by Kohler and Milstein, Nature256:495-497 (1975). The same preparation of the C5a(1-71,T1M,C27S,Q71C)antigen (coupled to KLH) was used to immunize mice. Screening ofmonoclonal cell lines generated by fusing spleen cells from immunizedmice with the hybridoma line P3/NSI/1-Ag4-1 (ATCC TIB 18) was carriedout using C5a(1-71,T1M,C27S,Q71C)/BSA and C5a/BSA. In a direct parallelto the procedure carried out with the polyclonal rabbit antisera, thoseantibodies which only reacted with C5a(1-71,T1M,C27S,Q71C) and not withC5a were used as specific monoclonal anti-C5a(1-71,T1M,C27S,Q71C)antibodies. Those which recognized both were used as detectionantibodies, and labelled with alkaline phosphatase.

EXAMPLE 16 Construction Of Gene Fusions Between Human Carbonic AnigdraseII (hCAII) Gene and C5A Receptor Antagonist Gene.

Plasmid pWCB401 contains a DNA sequence coding for the C5a receptorantagonist ((C5a(1-71, Thr1Met, Cys27Ser, Gln71Cys)). This plasmid wasprepared using Seq. ID No. 23 in the cassette mutagenesis proceduredescribed in Example 3. This plasmid then was modified by insertion of adouble-stranded synthetic oligonucleotide (OL1/OL2; OL1- 5'-AGC TGG GATCCG ATA TCC-3' (SEQ. ID No. 53), OL2 5'-A GCT GGA TAT CGG ATC CC-3'(SEQ. ID. No. 54)) at the unique HindIII restriction site of pWCB401using a standard ligation protocol (Maniatis et. al.) to yield plasmidpWCB401BE. This insertion added BamHI and EcoRV restriction sitesimmediately downstream of the stop codon and eliminated the HindIIIsite. Then, two more oligonucleotides (OL3/OL4; OL3--5'-A GCT TTC GTTGAC GAC GAC GAT AAA AAC GGT CTG CA-3'(SEQ. ID. No. 55), OL4--5 'G ACCGTT TTT ATC GTC GTC GTC AAC GAA-3'(SEQ. ID. No. 56)) were synthesized,phosphorylated and annealed by standard procedures (Maniatis et al.,1989). This synthesized oligonucleotide links the gene for the C5areceptor antagonist to the hCAII gene and encodes an enterokinaseprotease-, and an hydroxylamine sensitive cleavage site.

The 222 bp PstI-EcoRV fragment was recovered from pWcB401BE and wasligated in a three-way ligation experiment to the annealeddouble-stranded oligonucleotides OL3/OL4 and the HindIII and EcoRVdigested pBO304ΔRV. pBO304ΔRV is a derivative of pB0304 (Van Heeke etal., Protein Expression and Purification 4: 265-274 (1993)) and wasobtained by eliminating the EcoRV restriction site in the tetracyclineresistance gene. The product of the three-way ligation was designatedplasmid 29A-1, and th e DNA sequence surrounding the cloning junctionsincluding the synthetic oligonucleotides was verified by standard DNAsequencing methods (Maniatis et al.). E. coli strains HMS174(DE3)pLysSand BL21(DE3)pLysS both from (NOVAGEN, MADISON, WIS.) were transformedwith plasmid 29A-1 and colonies were isolated on Luria Broth agar platessupplemented with tetracycline (Maniatis et al.).

EXAMPLE 17A Construction Of C5A Fusion Proteins; Determination OfExpression TITERS in E. coli

In the course of experiments trying to exchange a fragment of the humaninterleukin-1B gene (modified with E. coli-preferred codons, obtainedfrom British Biotechnology Limited, UK) with a fragment coding for apeptide fragment of human interleukin-1 receptor antagonist (hIL1-RA) byoverlap extension PCR, an artifact translational stop codon followingthe codon for amino acid at position 72 was obtained. A BamHI cleavagesite (GGATCC) was later introduced at the stop codon site to facilitatethe construction of fusion proteins. The thus-obtained synethic gene isas follows:

    __________________________________________________________________________    DNA sequence of IL72 flanked by NcoI and GamHI                                restriction sites                                                             __________________________________________________________________________    5'CCATGGCACCGGTTAGATCTCTGAACTGCACCCTTCGCGACTCCCAACAG                          AAGAGCTTAGTAATGTCTGGTCCGTACGAGCTCAAAGCTCTGCATCTGCA                            AGGCCAGGACATGGAACAACAGGTTGTATTCAGCATGAGCTTCATTGAGC                            CTCATGCTCTTGCATTAGGCCTGAAAGAGAAGAATCTGTACCTCAGCTGC                            GTACTGAAAGCTGCGTCTCATATGTTGGATCC-3'(Seq. ID No. 57)                           __________________________________________________________________________

This synthetic gene is flanked by NcoI and BamHI sites and codes for ahybrid protein composed of sequences of human interleukin-1B (hIL-1B)and human interleukin-1 receptor antagonist (hILl-RA).

This synthetic gene was then cloned as a NcoI-BamHI fragment intoplasmid pPLMu resulting in plasmoid pPLMuIL72. Plasmid pPLMu is plasmidpPLmuSMCori (Buell, G. et al., Nucleic Acids Res. (1985) 13:1023-1038)with the NcoI-HindIII fragment replaced by a multiple cloning site setforth as below:

    __________________________________________________________________________    Sequence of EcoRI-Hind III fragment in                                        pPLmuSMCori containing the ribosome binding site and                          multiple cloning site                                                         __________________________________________________________________________    5'-GAATTCTTACACTTAGTTAAATTGCTAACTTTATAGATTACAAAACTTAG                         GAGGGTTTTTACCATGGTTACGAATTCCCGGGGATCCGTCGACCTGCAGC                            CAAGCTT-3'(Seq. ID No. 58)                                                    __________________________________________________________________________

The encoded hybrid protein is composed--counting from its N-terminalmethionine--of amino acids 1-47 of hIL-1B, followed by amino acids 52-57of hIL1-RA followed by amino acids 60-71 of hIL-1B, followed by theamino acid sequence Cys Val Leu Lys Ala Ala Ser (SEQ. ID. No. 59), and atranslational stop codon.

E. coil strain LC 137 (Goff, S. A. et al., Nat'l Proc. Acad. Sci., USA81:6647-6651 (1984)) was then transformed with plasmid pPLmulL72carrying the compatible plasmid pcl₈₅₇ encoding the thermolabile phageλCIS₅₇ repressor. Heat induction resulted in high expression of thehybrid protein as analyzed by SDS-PAGE of heat induced E. coli cells(Buell, et al., supra). The hybrid protein encoded by plasmid pPLMuIL72was further shortened by PCR using primers C, D and E as 3' primers:##STR14## as 5' primer resulting in plasmids pPLMulL33, pPLMulL53 andpPLMulL63, respectively. The sequence GAT (Asp) in all theseconstructions of the BamHI site is in proper reading frame with the DNAsequence coding for the hybrid protein. Any coding region cloned intothis site via BamHI cleavage will be preceeded by the acid labile aminoacid sequence Asp-Pro encoded by the sequence GATCCX which is partlycontained in the BamHI cleavage sequence. Acid cleavage of the thesefusion proteins will liberate the fusion partner through cleavage at theacid labile Asp-Pro site. The constructs in pPLMuIL63 and pPLMuIL53showed the same amount of hybrid protein expression as the originalpPLMuIL72. No expression was observed with pPLMuIL33. Expression levelswere determined afters SDS-PAGE and Coomassie® (ICI, Ltd.) BrilliantBlue staining. pPLMuIL33 and PPLMuIL53 were then used to constructfusion proteins with human C5a. A BamHI site coding for an asparagine(GAT) which is in proper reading frame with the following hC5a gene wasintroduced by primer directed PCR mutagenesis. The BamHI-HindIII-cut C5afragment was ligated into BamHI-HindIII cut plasmids pPLMuIL33 andpPLMuIL53 resulting in the plasmids pPLMuIL33-C5a and pPLMuIL53-C5a,respectively. ##STR15##

Heat induction of E. coli K12 LC 137 carrying pPLMuIL33-C5a andpPLMuIL53-C5a results in high expression of the fusion protein as judgedby SDS-PAGE and WESTERN analysis with anti-hC5a polyclonal antibodies.

EXAMPLE 17b Construction Of A Gene Fusion Between A Fragment Of A HumanInterleukin 1 Receptor Antagonist And C5a Analogue

The 5' region of plasmid pPLMmIL53-C5a encompassing codons 1-50 wasrecovered and optimized using PCR for the purpose of expressing a fusionprotein with C5aRA. Briefly, the 5' end was adapted to facilitatecloning into the pET-series bacterial expression vectors. The cysteinecodon at position 9 was replaced by a serine codon, and the 3' end ofthe fragment was modified to allow for the fusion with C5aRA andcleavage linker. The following experiments were performed.

The gene encoding the IL1RA fragment was recovered and adapted fromplasmid pPLMmIL53-C5a by PCR using standard DNA amplification conditions(Maniatis et al., 1989) and oligonucleotides designated G83 and G84 asprimers (G83: 5'-C GCA AGC TTG AGG CTC AAT GAA GCT CAT--3' (Seq. ID No.66)) (G84: 5'-GGA GAT ATA CAT ATG GCA CCG GTT AGA TCT CTG AAC AGC ACCCTT CGC-3'(SEQ ID No. 67)) . The amplified fragment was digested withrestriction endonucleases NdeI and HindIII and recovered from an agarosegel (fragment 1). Plasmid 29A-1 was digested with HindIII and BamHI andthe fragment containing the C5aRA gene was recovered (fragment 2). pET9c(pET System Manual, 3rd edition, 1993, Novagen) was digested with NdeIand BamHI and used in a three-way ligation experiment with fragments 1and 2 to construct plasmid 58A-l. In this construct, the gene coding forC5aRa was linked to a modified fragment of the hybrid IL1 gene fragmentvia a linker sequence that codes for an enterokinase and hydroxylaminecleavage site. E. coli strains BL21(DE3)pLysS and HMS174(DE3)pLysS weretransformed with p58A-1 and colonies were isolated on Luria Broth agarplates supplemented with kanamycin and chloramphenicol (Maniatis et al.,1989).

EXAMPLE 18 Expression Of C5a Analogue Fusion Proteins In E. coli

Expression experiments were performed using standard procedures for theT7/pET expression system described in Studier et al., Methods Enzymol.185:60-89 (1990), and Van Heeke et al., Protein Expression andPurification 4:265-274 (1993). Briefly, single E. coli coloniesharboring expression plasmid were grown in Luria Broth supplemented withthe appropriate antibiotic for selection until the optical density(measured at 550 min.) reached about 0.6. At this point, IPTG and Zn++were added to a final concentration of 0.4 mM and 12.5 μM, respectively.Incubation of cells was continued at either 21°, 30 ° or 37° C. for anadditional 2-4 hrs. Cells were harvested by centrifugation and stored at-80° C. until use.

EXAMPLE 19 Hydroxylamine Cleavage Of C5A Analogue Fusion Protein

Recombinant fusion protein was isolated from the frozen E. coli pastealiquots from Example 18 after thawing in a 50 mM Tris/HCI buffer,containing 1 mM EDTA at pH 8.0 (5:1 v:w, buffer:cell paste). The cellswere then disrupted by sonication, and the crude extract centrifuged for20 min at 3,000 ×g. The fusion protein-containing pellet was solubilizedin a solution containing 0.2 M CAPSO, 2 M NH₂ OH.HCL, 6 M guanidiniumhydrochloride, adjusted to pH 9.3 with LiOH (7:1 v:w, buffer:cellpellet).

The solution was incubated at 37° C. for 7 hours to promote fusionprotein cleavage by hydroxylamine to occur in excess of 80%, after whichthe pH was adjusted to 8.0 with 4 M HCl. The solution, containing inexcess of 80% hydroxylamine cleaved fusion protein, was dialyzed against25 mM Tris.HCL at pH 7.3 overnight. The dialysate was then centrifugedat 30,000 ×g for 20 min and the C5aRA-containing precipitate collected.The precipitate was solubilized in 6 M guanidinium hydrochloride (5:1v:w, buffer:precipitate), and diluted twenty-fold with 100 mM Tris/HClbuffer at pH 7.4 containing 1 mM reduced/0.01 mM oxidized glutathione asdescribed in Example 5b.

Purification and dimerization were then performed according to themethods as described above.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All these publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

Various modifications of the invention described herein will becomeapparent to those skilled in the art. Such modifications are intended tofall within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 67                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TyrAspGlyAla                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AspGlyAlaTyr                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 74 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ThrLeuGlnLysLysIleGluGluIleAlaAlaLysTyrLysHisSer                              151015                                                                        ValValLysLysCysCysTyrAspGlyAlaCysValAsnAsnAspGlu                              202530                                                                        ThrCysGluGlnArgAlaAlaArgIleSerLeuGlyProArgCysIle                              354045                                                                        LysAlaPheThrGluCysCysValValAlaSerGlnLeuArgAlaAsn                              505560                                                                        IleSerHisLysAspMetGlnLeuGlyArg                                                6570                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 239 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AATTCTATGACTCTGCAAAAGAAGATCGAAGAAATCGCTGCTAAGTACAAGCACTCCGTC60                GTTAAGAAGTGTTGTTACGATGGTGCATGCGTCAACAACGACGAAACCTGTGAACAACGA120               GCTGCTCGTATTTCTCTGGGCCCTCGCTGTATCAAGGCTTTCACTGAATGTTGTGTTGTC180               GCTTCCCAACTGCGCGCTAACATTTCTCACAAGGACATGCAACTCGGCCGCTAAAAGCT239                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AsnIleSerHisLysAspMetGlnLeuGlyArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ACGGTGCTTCTGTTAACA18                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CTGCGTGCTAACATCTCTCACAAAGACATGTGCTA35                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AGCTTAGCACATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CTGCGTGCTTGCTA14                                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AGCTTAGCAAGCACGCAG18                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CTGCGTGCTAACTGCTA17                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AGCTTAGCAGTTAGCACGCAG21                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CTGCGTGCTAACATCTGCTA20                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AGCTTAGCAGATGTTAGCACGCAG24                                                    (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CTGCGTGCTAACATCTCTTGCTA23                                                     (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AGCTTAGCAAGAGATGTTAGCACGCAG27                                                 (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CTGCGTGCTAACATCTCTCACTGCTA26                                                  (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AGCTTAGCAGTGAGAGATGTTAGCACGCAG30                                              (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CTGCGTGCTAACATCTCTCACAAATGCTA29                                               (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      AGCTTACGATTTGTGAGAGATGTTAGCACGCAG33                                           (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CTGCGTGCTAACATCTCTCACAAAGACTGCTA32                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AGCTTAGCAGTCTTTGTGAGAGATGTTAGCACGCAG36                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CTGCGTGCTAACATCTCTCACAAAGACATGTGCTA35                                         (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AGCTTAGCACATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CTGCGTGCTAACATCTCTCACAAAGACATGCAATGCTA38                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AGCTTAGCATTGCATGTCTTTGTGAGAGATGTTAGCACGCAG42                                  (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CTGCGTGCTAACATCTCTCACAAAGACATGCAACTGTGCTA41                                   (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      AGCTTAGCACAGTTGCATGTCTTTGTGAGAGATGTTAGCACGCAG45                               (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CTGCGTGCTAACATCTCTCACAAAGACATGTGCCTGGGTCGTTA44                                (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AGCTTAACGACCCAGGCACATGTCTTTGTGAGAGATGTTAGCACGCAG48                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CTGCGTGCTAACATCTCTCACAAAGACATGTGCCTGGGTTA41                                   (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      AGCTTAACCCAGGCACATGTCTTTGTGAGAGATGTTAGCACGCAG45                               (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CTGCGTGCTAACATCTCTCACAAAGACATGTGCCTGTA38                                      (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      AGCTTACAGGCACATGTCTTTGTGAGAGATGTTAGCACGCAG42                                  (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CTGCGTGCTAACATCTCTCACAAAGACATGCAACTGGGTTGCTA44                                (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AGCTTAGCAACCCAGTTGCATGTCTTTGTGAGAGATGTTAGCACGCAG48                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      CTGCGTGCTAACATCTCTCACAAAGACATGCAATA35                                         (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      AGCTTATTGCATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CTGCGTCCTAACATCTCTCACAAAGACATGGACTA35                                         (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      AGCTTAGTCCATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      CTGCGTGCTAACATCTCTCACAAAGACATGTCTTA35                                         (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AGCTTAAGACATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CTGCGTGCTAACATCTCTCACAAAGACATGCACTA35                                         (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AGCTTAGTGCATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      CTGCGTGCTAACATCTCTCACAAAGACATGCGTTA35                                         (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      AGCTTAACGCATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      CTGCGTGCTAACATCTCTCACAAAGACATGCTGTA35                                         (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AGCTTACAGCATGTCTTTGTGAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      CTGCGTGCTAACATCTCTTTCAAAGACATGTGCTA35                                         (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      AGCTTAGCACATGTCTTTGAAAGAGATGTTAGCACGCAG39                                     (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 252 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      GAATTCCCACTCAAAATAAGGAGGAAAAAAAAATGCTGCAGAAGAAAATCGAAGAAATCG60                CTGCTAAGTACAAACACTCTGTTGTTAAAAAATGCTGCTACGACGGTGCTTCTGTTAACA120               ACGACGAAACTTGCGAACAGCGTGCTGCTCGTATCTCTCTGGGCCCGCGTTGCATCAAAG180               CATTCACTGAATGCTGCGTTGTTGCTTCTCAGCTGCGTGCTAACATCTCTTTCAAAGACA240               TGTGCTAAGCTT252                                                               (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      IleSerPheLysAspMetGlnLeuGlyArg                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      AGCTGGGATCCGATATCC18                                                          (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      AGCTGGATATCGGATCCC18                                                          (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      AGCTTTCGTTGACGACGACGATAAAAACGGTCTGCA36                                        (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      GACCGTTTTTATCGTCGTCGTCAACGAA28                                                (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 232 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      CCATGGCACCGGTTAGATCTCTGAACTGCACCCTTCGCGACTCCCAACAGAAGAGCTTAG60                TAATGTCTGGTCCGTACGAGCTCAAAGCTCTGCATCTGCAAGGCCAGGACATGGAACAAC120               AGGTTGTATTCAGCATGAGCTTCATTGAGCCTCATGCTCTTGCATTAGGCCTGAAAGAGA180               AGAATCTGTACCTCAGCTGCGTACTGAAAGCTGCGTCTCATATGTTGGATCC232                       (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      GAATTCTTACACTTAGTTAAATTGCTAACTTTATAGATTACAAAACTTAGGAGGGTTTTT60                ACCATGGTTACGAATTCCCGGGGATCCGTCGACCTGCAGCCAAGCTT107                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      CysValLeuLysAlaAlaSer                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      CTTATAGGATCCAGATTCTTCTCTTTCAGGCCTAATGCAAG41                                   (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      CTTATAGGATCCAGAGCATGAGGCTCAATGAAG33                                           (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      CTTATAGGATCCAGATGCAGAGCTTTGAGCTC32                                            (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      TATAAGTCCATGGCACCGGTTAG23                                                     (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 316 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      CCATGGCACCGGTTAGATCTCTGAACTGCACCCTTCGCGACTCCCAACAGAAGAGCTTAG60                TAATGTCTGGTCCGTACGAGCTCAAAGCTCTGCATCTGGATCCCTGCAGAAGAAAATCGA120               AGAAATCGCTGCTAAGTACAAACACTCTGTTGTTAAAAAATGCTGCTACGACGGTGCTTC180               TGTTAACAACGACGAAACTTGCGAACAGCGTGTCGCTCGTATCTCTCTGGGCCCGCGTTG240               CATCAAAGCATTCACTGAATGCTGCGTTGTTGCTTCTCAGCTGCGTGCTAACATCTCTCA300               CAAAGACATGTGCTAA316                                                           (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 379 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      CCATGGCACCGGTTAGATCTCTGAACTGCACCCTTCGCGACTCCCAACAGAAGAGCTTAG60                TAATGTCTGGTCCGTACGAGCTCAAAGCTCTGCATCTGCAAGGCCAGGACATGGAACAAC120               AGGTTGTATTCAGCATGAGCTTCATTGAGCCTCATGCTCTGGATCCCTGCAGAAGAAAAT180               CGAAGAAATCGCTGCTAAGTACAAACACTCTGTTGTTAAAAAATGCTGCTACGACGGTGC240               TTCTGTTAACAACGACGAAACTTGCGAACAGCGTGTCGCTCGTATCTCTCTGGGCCCGCG300               TTGCATCAAAGCATTCACTGAATGCTGCGTTGTTGCTTCTCAGCTGCGTGCTAACATCTC360               TCACAAAGACATGTGCTAA379                                                        (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      CGCAAGCTTGAGGCTCAATGAAGCTCAT28                                                (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      GGAGATATACATATGGCACCGGTTAGATCTCTGAACAGCACCCTTCGC48                            __________________________________________________________________________

We claim:
 1. A DNA molecule encoding a polypeptide analogue of humanC5a, wherein said analogue is a C5a receptor antagonist that exhibitssubstantially no agonist activity, and wherein said analogue comprises aC-terminal region which differs from the corresponding C-terminal regionof human C5a in that it is truncated by at least two amino acidresidues, and contains at least one cysteine residue, provided that theC-terminal amino acid of said region is a cysteine residue, and whereinsaid analogue comprises an N-terminal region comprising C5a(2-63) or atleast one fragment or a conservatively substituted variant thereof.
 2. ADNA molecule encoding a polypeptide analogue of human C5a,comprising:(a) an N-tennlnal region comprising C5a (2-63), and (b) aC-terminal region which differs from the corresponding C-terminal regionof C5a in that said C-terminal region is truncated by at least two aminoacid residues, and wherein said region contains at least one cysteineresidue, provided that the C-terminal amino add of aid region iscysteine, and wherein said polypeptide analogue is a C5a receptorantagonist that exhibits substantially no antagonist activity.
 3. TheDNA molecule of claim 2 or claim 1, wherein said N-terminal region ofsaid analogue comprises C5a (1-63,Thr1Met).
 4. The DNA molecule of claim2 or claim 1, which encodes C5a (1-71, Thr1Met, Gln71Cys).
 5. The DNAmolecule of claim 2 or claim 1, which encodes C5a (1-71, Thr1Met,His67Phe, Gln71Cys).
 6. the DNA molecule of claim 2 or claim 1, whereinsaid N-terminal region of said analogue comprised C5a (1-63, Thr1Gly).7. A DNA molecule encoding a polypeptide analogue of human C5a,comprising:(a) an N terminal region comprising C5a (2-63, Cys27Ser), and(b) a C-terminal region which differs from the corresponding C-terminalregion of C5a in that said C-terminal region is truncated by at leasttwo amino acid residues, and said region contains at least one cysteineresidue, provided that the C-terminal amino acid of said region iscysteine, and wherein said polypeptide analogue is a C5a receptorantagonist that exhibits substantially no agonist activity.
 8. The DNAmolecule of claim 7 or claim 1, which encodes C5(1-71, Thr1Met,Cys27Ser, Gln71Cys).
 9. The DNA molecule of claim 7 or claim 1, whichencodes C5a (1-71, Thr1Gly, Cys27Ser, His67Phe, Gln71Cys).
 10. The DNAmolecule of claim 7 or claim 1, which encodes C5a (1-71, Thr1Gly,Cys27Ser, His 67Phe, Gln71Cys).
 11. The DNA molecule of any one ofclaims 1, 2 or 7, wherein said analogue is from 64 to 72 amino acids inlength.
 12. The DNA molecule of any one of claims 1, 2, or 7, whereinsaid analogue is from 64 to 72 amino acids in length.
 13. The DNAmolecule of any one of claims 1, 2, or 3, wherein said analogue is from68 to 72 amino acids in length.
 14. The DNA molecule of any one ofclaims 1, 2, or 3, wherein said analogue is from 70 to 72 amino acids inlength.
 15. The DNA molecule of any one of claims 1, 2, or 3, whereinsaid analogue is 71 amino acids in length.
 16. A recombinant DNAmolecule, comprising a promoter capable of functioning in a given hostoperably linked to the DNA molecule of any one of claims 1, 2, or
 3. 17.A recombinant plasmid compatible with a given host, comprising therecombinant DNA molecule of claim
 16. 18. A recombinant vectorcompatible with a given host, comprising the recombinant DNA molecule ofclaim
 16. 19. A recombinant host cell stably transformed with therecombinant DNA molecule of claim
 16. 20. The recombinant host of claim19, selected from the group consisting of bacterial, yeast, fungal,insect, mammalian and plant cells.
 21. The recombinant host cell ofclaim 19, which is E. coli.
 22. The recombinant host cell of claim 19,wherein said DNA molecule encodes C5a (1-71, Thr1Met, Cys27Ser,Gln71Cys).
 23. The recombinant host cell of claim 19, wherein said DNAmolecule encodes C5a (1-71, Thr1Gly, Cys27Ser, Gln71Cys).
 24. A methodof preparing a biologically active human C5a analogue, wherein saidanalogue is a C5a receptor antagonist that exhibits substantially noagonist activity, and wherein said analogue comprises a C-terminalregion which differs from the corresponding C-terminal region of humanC5a in that it is truncated by at least two amino acid residues, andcontains at least one cysteine residue, provided that the C-terminalamino acid of said region is a cysteine residue, and wherein saidanalogue comprises an N-terminal region comprising C5a(2-63) or leastone fragment or a conservatively substituted variant thereof, comprisingthe steps of:culturing E. coli cells stably transformed with a DNAmolecule encoding the C5a analogue under conditions suitable to causeexpression of the DNA molecule; contacting the thus-cultured cells witha denaturing and solubilizing agent to produce the c5a analogue indenatured form; and mixing the thus denatured C5a analogue with asolution containing a reducing agent and an oxidizing agent in a molarratio of the reducing agent to the oxidizing agent by, weight of atleast about 100:1 under suitable conditions to produce the C5a analoguein biologically active form.
 25. The method of claim 24, wherein saidmixing is conducted at a pH of from about 6.5 to about 7.5.
 26. Themethod of claim 24, wherein said mixing is conducted from about 1/2 hourto about 4 hours.
 27. The method of claim 24, wherein said the redoxcouple is reduced glutathione/oxidized glutathione.
 28. The method ofclaim 24, wherein said DNA molecule encodes the C5a analogue in the formof a fusion protein.
 29. The method of claim 28, further comprising thestep of cleaving the thus-expressed fusion protein prior to said step ofmixing.
 30. The method of claim 24, wherein DNA molecule encodes apolypeptide analogue of human C5a,(a) wherein said N-terminal region ofsaid analogue comprised C5a (2-63).
 31. The method of claim 30, whereinsaid N-terminal region of said analogue comprises C5a (1-63, Thr1Met).32. The method of claim 30, wherein said DNA molecule encodes C5a (1-71,Thr1Met, Gln71Cys).
 33. The method of claim 30, wherein said DNAmolecule encodes C5a (1-71, Thr1Met, His 67Phe, Gln71Cys).
 34. Themethod of claim 30, wherein said N-terminal region of said analoguecomprises C5a (1-63, Thr1Gyn).
 35. The method of claim 24, wherein saidDNA molecule encodes a polypeptide analogue of human C5a,(a) whereinsaid N-terminal region of said analogue comprises C5a (2-63) (Cys27Ser).36. The method of claim 35, wherein said DNA molecule encodes C5a (1-71,Thr1Met, Cys27Ser, Gln71Cys).
 37. The method of claim 35, wherein saidDNA molecule encodes C5a (1-71, Thr1Met, Cys27Ser, His67Phe, Gln71Cys).38. The method of claim 24, wherein said analogue has a glycine residueas the N-terminus.
 39. The method of claim 24, wherein said DNA moleculeencodes C5a (1-71, Thr1Gly, Cys27Ser, Gln71Cys).
 40. The method of claim24, wherein said DNA molecule encodes C5a (1-71, Thr1Gly, Cys27Ser,His67Phe, Gln71Cys).
 41. A method of preparing a biologically activehuman C5a analogue, wherein said analogue is a C5a receptor antagonistthat exhibits substantially no agonist activity, and wherein saidanalogue comprises a C-terminal region which differs from thecorresponding C-terminal region of human C5a in that it is truncated byat least two amino acid residues, and contains at least one cysteineresidue, provided that the C-terminal amino acid of said region is acysteine residue, and wherein said analogue comprises an N-terminalregion comprising C5a(2-63) or at least one fragment or a conservativelysubstituted variant thereof, comprising the steps of:culturing hostcells stably transformed with a recombinant DNA molecule encoding theC5a analogue in the form of a fusion protein wherein said culturing isconducted under conditions suitable to cause expression of said fusionprotein; isolating the fusion protein from the thus-cultured host cells;and cleaving the thus-isolated fusion protein so that the C5a analoguecan be obtained.
 42. The method of claim 41, wherein said host cells areE. coli cells.
 43. The method of claim 41, wherein said analogue has aglycine residue as the N-terminus.
 44. The method of claim 41, whereinsaid recombinant DNA molecule encodes C5a (1-71, Thr1Gly, Cys27Ser,Gln71Cys).
 45. The method of claim 41, wherein said recombinant DNAmolecule encodes C5a (1-71, Thr1Gly, Cys27Ser, His67Phe, Gln71Cys).